Nicotine Immunonanotherapeutics

ABSTRACT

The present invention provides compositions and systems for delivery of nanocarriers to cells of the immune system. The invention provides nanocarriers capable of stimulating an immune response in T cells and/or in B cells. The invention provides nanocarriers that comprise an immunofeature surface having a plurality of nicotine moieties. The invention provides pharmaceutical compositions comprising inventive nanocarriers. The present invention provides methods of designing, manufacturing, and using inventive nanocarriers and pharmaceutical compositions thereof. For example, the present invention nanocarriers capable of eliciting an immune response and the production of anti-nicotine antibodies.

RELATED APPLICATIONS

This application is a continuation-in-part of PCT Application Serial No.PCT/US08/011,932, filed Oct. 12, 2008, the entire contents of which isherein incorporated by reference.

GOVERNMENT SUPPORT

This invention was made with government support under Grant Nos.EB003647, AI072252, and AI069259, awarded by the NIH. The government hascertain rights in this invention.

BACKGROUND OF THE INVENTION

Many current vaccines against microbial pathogens comprise liveattenuated or non-virulent strains of the causative microorganisms. Manyvaccines comprise killed or otherwise inactivated microorganisms. Othervaccines utilize purified components of pathogen lysates, such assurface carbohydrates or recombinant pathogen-derived proteins. Vaccinesthat utilize live attenuated or inactivated pathogens typically yield avigorous immune response, but their use has limitations. For example,live vaccine strains can sometimes cause infectious pathologies,especially when administered to immune-compromised recipients. Moreover,many pathogens, particularly viruses, undergo continuous rapid mutationsin their genome, which allow them to escape immune responses toantigenically distinct vaccine strains.

Given the difficulty of vaccine development, many vaccines are inextremely short supply. For example, as of October 2007, there areinfluenza, varicella, and hepatitis A vaccine shortages in the UnitedStates. In some instances, vaccine shortages occur because not enoughmanufacturers devote their facilities to vaccine production to keep upwith demand. In some cases, vaccine shortages are attributed to lowpotency of the vaccine, which means a large amount of vaccine productmust be administered to each individual in order to achieve aprophylactic effect. For example, some vaccines cannot be administeredas an intact organism (even if attenuated or killed) because they causeinfectious pathologies. Instead, such vaccines usually comprise purifiedpathogen components, which typically leads to a much less potent immuneresponse.

Thus, there is a need in the art for systems and methods for producinghighly immunogenic, effective vaccines. There is also a need forimproved vaccine compositions that can potently induce long-lastingimmune responses. For the treatment and prevention of infectiousdiseases, there is a need for improved vaccine compositions that arehighly immunogenic but do not cause disease.

Smoking of cigarettes, cigars, and pipes is a prevalent problem in theUnited States and worldwide. Smoking tobacco and smokeless tobacco arerich in nicotine, which is a known addictive substance. Peak levels ofnicotine in the blood, about 25 to 50 nanograms/ml, are achieved within10-15 minutes of smoking a cigarette. In humans, smoking a cigaretteresults in arterial nicotine concentrations being 10-fold higher thanvenous nicotine concentrations because nicotine is rapidly deliveredfrom the lungs to the heart (see Henningfield (1993) Drug AlcoholDepend. 33:23-29). This results in a rapid delivery of high arterialconcentrations of nicotine to the brain. Once nicotine crosses theblood-brain barrier, evidence suggests that it binds to cholinergicreceptors. When nicotine binds to these receptors, it can affect normalbrain function, by triggering the release of other neurotransmitters,such as dopamine Dopamine is found in the brain in regions involved inemotion, motivation, and feelings of pleasure. It is the release ofneurotransmitters, especially dopamine, that is responsible for thetobacco user's addiction to nicotine or other intake of nicotine.

Nicotine is an alkaloid derived from the tobacco plant that isresponsible for smoking's psychoactive and addictive effects. Nicotineis formed of two rings linked together by a single bond: an aromaticsix-membered ring (pyridine) and an aliphatic five-membered ring(pyrrolidine). The pyrrolidine is N-methylated and linked through itscarbon-2 to the carbon-3 of pyridine. Thus, the carbon-2 is chiral, andthere is virtually free rotation around the single bond linking the tworings. It has been established that the absolute configuration ofcarbon-2 is S. Thus, the natural configuration of nicotine is(S)-(−)-nicotine.

Therapies for nicotine addiction have been developed, but are largelyineffective. The two most popular therapies remain the nicotinetransdermal patch and nicotine incorporated into chewing gum. Thesetherapies, termed “nicotine replacement therapies” (NRTs), replace theamount of nicotine which the user previously received from smoking andact to wean the user off nicotine. However, certain drawbacks are seenwith this type of therapy. Particularly, there is low penetration ofnicotine into the bloodstream and therefore an increased desire tosmoke.

There remains a need in the art to develop improved methods of treatingaddiction (such as addictions to nicotine, cocaine, heroine, alcohol,and other drugs). Ideal methods for treating addiction would, forexample, result in minimal withdrawal symptoms, encourage patientcompliance by being simple to administer, and result in low relapserates among patients.

SUMMARY OF THE INVENTION

The present invention provides synthetic nanocarriers for modulating theimmune system. The synthetic nanocarriers comprise one or more surfaces,where at least one of the surfaces comprises an immunofeature surface.Optionally the synthetic nanocarriers of the invention further containone or more of an immunomodulatory agent, an immunostimulatory agent,and a targeting agent (also referred to herein as “targeting moiety”).The immunomodulatory agent induces an immune response in B and/or Tcells. The immunostimulatory agent helps stimulate the immune system (ina manner that can ultimately enhance, suppress, direct, or redirect animmune response). The immunofeature surface recognizes one or moretargets associated with antigen presenting cells. The optional targetingagent recognizes one or more targets associated with a particular organ,tissue, cell, and/or subcellular locale. The nanocarriers are useful inpharmaceutical preparations and kits for the prophylaxis and/ortreatment of diseases, disorders, or conditions susceptible to treatmentby immune system modulation. Such conditions include those diseases,disorders, or conditions modified by enhancing the immune responsespecifically or nonspecifically, suppressing the immune responsespecifically or nonspecifically, or directing/redirecting the immuneresponse specifically or nonspecifically.

An immunofeature surface, as described in more detail herein, providesfor specific targeting of the nanocarriers to antigen presenting cells(APCs). In particular, the immunofeature surface provides for highavidity binding of the nanocarriers to APC surfaces. Furthermore, thehigh avidity binding is specific to APC cells. For example, in someembodiments, nanocarriers of the invention are capable of specificallytargeting subcapsular sinus macrophages (SCS-Mphs). Such nanocarriersaccumulate in the subcapsular sinus region of lymph nodes whenadministered to a subject. In other embodiments, the nanocarriers of theinvention are capable of specifically targeting dendritic cells andeliciting a T-cell response. In some preferred embodiments, theimmunofeature surface provides low affinity, high avidity binding of thenanocarriers to APC surfaces. In some embodiments, nanocarrierscomprising an immunofeature surface exhibit specific low affinity highavidity binding to APCs, and do not provide such binding to other typesof cells Further details of immunofeature surfaces are provided herein.

The ability of the immunofeature surface to target APCs is a key featurethat allows the nanocarriers of the invention to deliverimmunostimulatory and immunomodulatory agents to B-cells and/or T-cellswhen administered to a subject. Such delivery allow the inventivenanocarriers to elicit an immune system response, or to enhance animmune system response.

For example, in some embodiments, the immunofeature surface comprisesnicotine moieties.

As will be recognized by those skilled in the art, immune systemmodulation is useful, among other things, in connection with medicaltreatments, such as, for example, for prophylaxis and/or treatment ofinfectious disease, cancer, autoimmune disease (including rheumatoidarthritis), immune suppression in connection with transplants toameliorate transplant rejection, immunization against addictivesubstances, and immunization against biohazards and other toxicsubstances. Immune system modulation also is useful as a tool inindustrial and academic research settings, such as, for example, toimmunize an animal to produce antibodies.

One aspect of the invention is the provision of vaccines. A vaccineaccording to the invention typically contains an antigen. In oneembodiment, the antigen is physically ‘bound’ to the nanocarrier bycovalent or noncovalent means. Noncovalently bound includes, forexample, ionic bonding, hydrophobic bonding, physical entrapment, andthe like, all described in greater detail below. Such nanocarriers whichthemselves carry an antigen are included in the category referred tobelow as vaccine nanocarriers. In another embodiment, the nanocarrierhas bound to it an immunostimulatory agent for enhancing, suppressing,directing, or redirecting an immune response, preferably to an antigen.In this case, the antigen may be mixed with the preparation of agentbound nanocarrier to which the immunostimulatory agent is bound form thevaccine. The antigen, of course may also be bound to a nanocarrier,including as discussed below, the same nanocarrier to which theimmunostimulatory agent is bound. The antigen may also be a moiety ofthe immunofeature surface.

The preparations of the invention in many instances will include one ormore nanocarriers. In some embodiments, the preparation includes ananocarrier having an immunofeature surface where the nanocarrier isbound to one or more, but not all, of an immunomodulatory agent, animmunostimulatory agent, and a targeting agent. In some embodiments, thepreparation is a mixture of nanocarriers with subpopulations carryingone or more, but not all, of an immunomodulatory agent, animmunostimulatory agent, and a targeting agent. In some embodiments, thepreparation is a mixture of different nanocarriers, each nanocarriercarrying one or more, but not all, of an immunomodulatory agent, animmunostimulatory agent, and a targeting agent. The preparationslikewise may be one of nanocarriers, wherein each nanocarrier has boundto it all of an immunomodulatory agent, an immunostimulatory agent, anda targeting agent. In this instance, the nanocarriers themselves, apartfrom the agents they deliver, may be the same or different. Thetargeting agents mentioned here (and as described in more detail herein)are, for example, B-cell targeting moieties or T-cell targetingmoieties. It will be appreciated that, throughout this disclosure, suchmoieties are in addition to the plurality of moieties that are presenton the immunofeature surface and that provide targeting of thenanocarriers to APCs.

Important is the discovery that the nanocarriers of the invention arepowerful at stimulating the immune system. Important is the discoverythat the nanocarriers can be fashioned to mimic, and from animmunological standpoint, improve on, what the immune system ‘sees’ whenexposed to antigens in nature or in prior vaccine technology. In thisrespect, it has been discovered unexpectedly that the activity ofadjuvants can be markedly enhanced if covalently bound to nanocarriers.It also has been discovered unexpectedly that the immunofeature surfaceof the nanocarriers can help target an immunomodulatory agent orimmunostimulatory agent to appropriate immune cells even without aspecific cell targeting agent.

The systems described herein permit the manipulation of the parametersaffecting the immune system in a manner which results in improved immunemodulation. One important aspect of the invention is that thenanocarriers can be controlled in terms of size, density of agent,degree and location of targeting, degradation, release of agent, etc. Avariety of aspects of the invention achieve one or more of thesebenefits, described in more detail below. In particular, below aredescribed immune modulating preparations, synthetic nanocarriercomponents of such preparations, specific and preferred nanocarriers,specific and preferred immunomodulatory, immunostimulatory, andtargeting agents, component parts and building blocks of nanocarriers ofthe invention, as well as methods for manufacturing such nanocarriers,including a preferred method involving self-assembled nanocarriers. Inaddition, preparations and systems for generating robust immunemodulation in connection with weak antigens and antigens not recognizedby T cells (e.g., carbohydrate and small molecule antigens) aredescribed. In some aspects, a composition comprising a nanocarrier(e.g., one that targets a specific organ, tissue, cell, or subcellularlocale) is provided. In some embodiments, the nanocarrier targets one ormore secondary lymphoid tissues or organs. In some embodiments, thesecondary lymphoid tissue or organ is the lymph nodes, spleen, Peyer'spatches, appendix, or tonsils.

The scaffold of the nanocarrier (and which the agents provided hereinmay be associated with or encapsulated by) can be composed of polymerand/or non-polymer molecules. Accordingly, the nanocarrier scaffold canbe protein-based, nucleic acid based, or carbohydrate-based. Thescaffold, in some embodiments, is macromolecular. In some embodiments,the scaffold is composed of amino acids or nucleic acids. In someembodiments, the scaffold is composed of crosslinking chains ofmolecules, such as nucleic acids. In some embodiments, the scaffold iscomposed of RNAi crosslinking chains. In some embodiments, the scaffoldis polyamino-based. A nanocarrier can be, but is not limited to, one ora plurality of lipid-based nanoparticles, polymeric nanoparticles,metallic nanoparticles, surfactant-based emulsions, dendrimers, and/ornanoparticles that are developed using a combination of nanomaterialssuch as lipid-polymer nanoparticles.

In some embodiments, the nanocarrier is composed of one or morepolymers. In some embodiments, the one or more polymers is a watersoluble, non-adhesive polymer. In some embodiments, polymer ispolyethylene glycol (PEG) or polyethylene oxide (PEO). In someembodiments, the polymer is polyalkylene glycol or polyalkylene oxide.In some embodiments, the one or more polymers is a biodegradablepolymer. In some embodiments, the one or more polymers is abiocompatible polymer that is a conjugate of a water soluble,non-adhesive polymer and a biodegradable polymer. In some embodiments,the biodegradable polymer is polylactic acid (PLA), poly(glycolic acid)(PGA), or poly(lactic acid/glycolic acid) (PLGA). In some embodiments,the nanocarrier is composed of PEG-PLGA polymers.

In some embodiments, the nanocarrier is formed by self-assembly.Self-assembly refers to the process of the formation of a nanocarrierusing components that will orient themselves in a predictable mannerforming nanocarriers predictably and reproducibly. In some embodiments,the nanocarriers are formed using amphiphilic biomaterials which orientthemselves with respect to one another to form nanocarriers ofpredictable dimension, constituents, and placement of constituents.According to the invention, the amphiphilic biomaterials may haveattached to them immunomodulatory agents, immunostimulatory agentsand/or targeting agents such that when the nanocarriers self assemble,there is a reproducible pattern of localization and density of theagents on/in the nanocarrier.

In some embodiments, the nanocarrier is a microparticle, nanoparticle,or picoparticle. In some embodiments, the microparticle, nanoparticle,or picoparticle is self-assembled.

In some embodiments, the nanocarrier has a positive zeta potential. Insome embodiments, the nanocarrier has a net positive charge at neutralpH. In some embodiments, the nanocarrier comprises one or more aminemoieties at its surface. In some embodiments, the amine moiety is aprimary, secondary, tertiary, or quaternary amine. In some embodiments,the amine moiety is an aliphatic amine. In some embodiments, thenanocarrier comprises an amine-containing polymer. In some embodiments,the nanocarrier comprises an amine-containing lipid. In someembodiments, the nanocarrier comprises a protein or a peptide that ispositively charged at neutral pH. In some embodiments, the nanocarrieris a latex particle. In some embodiments, the nanocarrier with the oneor more amine moieties on its surface has a net positive charge atneutral pH.

The nanocarriers of the compositions provided herein, in someembodiments, have a mean geometric diameter that is less than 500 nm. Insome embodiments, the nanocarriers have mean geometric diameter that isgreater than 50 nm but less than 500 nm. In some embodiments, the meangeometric diameter of a population of nanocarriers is about 60 nm, 75nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, or 475 nm. In someembodiments, the mean geometric diameter is between 100-400 nm, 100-300nm, 100-250 nm, or 100-200 nm. In some embodiments, the mean geometricdiameter is between 60-400 nm, 60-350 nm, 60-300 nm, 60-250 nm, or60-200 nm. In some embodiments, the mean geometric diameter is between75-250 nm. In some embodiments, 30%, 40%, 50%, 60%, 70%, 80%, 90%, ormore of the nanocarriers of a population of nanocarriers have a diameterthat is less than 500 nm. In some embodiments, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or more of the nanocarriers of a population ofnanocarriers have a diameter that is greater than 50 nm but less than500 nm. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or more of the nanocarriers of a population of nanocarriers have adiameter of about 60 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm,225 nm, 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm,450 nm, or 475 nm. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or more of the nanocarriers of a population ofnanocarriers have a diameter that is between 100-400 nm, 100-300 nm,100-250 nm, or 100-200 nm. In some embodiments, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or more of the nanocarriers of a population ofnanocarriers have a diameter that is between 60-400 nm, 60-350 nm,60-300 nm, 60-250 nm, or 60-200 nm. In some of the foregoingembodiments, the nanocarriers are nanoparticles.

The nanocarrier provided herein can be used to modulate an immuneresponse (e.g., enhance, suppress, direct, or redirect) and comprises animmunofeature surface. The nanocarriers may also comprise at least oneof an immunomodulatory agent, an immunostimulatory agent, and atargeting agent. In some embodiments, the nanocarrier comprises at leastone of a B cell antigen, a T cell antigen, an immunostimulatory agent,and a targeting agent. In some embodiments, the nanocarrier comprises atleast two of a B cell antigen, a T cell antigen, an immunostimulatoryagent, and a targeting agent. In some embodiments, the nanocarriercomprises at least three of a B cell antigen, a T cell antigen, animmunostimulatory agent, and a targeting agent. In some embodiments, thenanocarrier comprises all of a B cell antigen, a T cell antigen, animmunostimulatory agent, and a targeting agent.

In some embodiments, the nanocarrier comprises a B cell antigen. The Bcell antigen may be on the immunofeature surface of the nanocarrier, ona second surface of the nanocarrier, encapsulated within thenanocarrier, or combination thereof. In some embodiments, the B cellantigen is on the surface of the nanocarrier at a density whichactivates B cell receptors. In some embodiments, the B cell antigen isassociated with the nanocarrier. In some embodiments, the B cell antigenis covalently associated with the nanocarrier. In some embodiments, theB cell antigen is non-covalently associated with the nanocarrier. Insome embodiments, the nanocarrier further comprises a targeting moiety.In some embodiments, the B cell antigen is a poorly immunogenic antigen.In some embodiments, the B cell antigen is a small molecule. In someembodiments, the B cell antigen is an addictive substance. In someembodiments, the B cell antigen is a toxin. In some embodiments, thetoxin for inclusion in a nanocarrier is the complete molecule or aportion thereof. In some embodiments the B cell antigen is not a T cellantigen. In some embodiments, the B cell antigen is a carbohydrate. Insome embodiments, the B cell antigen is a degenerative disease antigen,an infectious disease antigen, a cancer antigen, an atopic diseaseantigen, an autoimmune disease antigen, an alloantigen, a xenoantigen,an addictive substance, or a metabolic disease enzyme or enzymaticproduct.

In some embodiments, the nanocarrier comprises a T cell antigen. In someembodiments, the T cell antigen is on the immunofeature surface of thenanocarrier, on a second surface of the nanocarrier, encapsulated withinthe nanocarrier, or combination thereof. In some embodiments, the T cellantigen is associated with the nanocarrier. In some embodiments, the Tcell antigen is covalently associated with the nanocarrier. In someembodiments, the T cell antigen is non-covalently associated with thenanocarrier. In some embodiments, the antigen is a degenerative diseaseantigen, an infectious disease antigen, a cancer antigen, an atopicdisease antigen, an autoimmune disease antigen, an alloantigen, axenoantigen, an addictive substance, or a metabolic disease enzyme orenzymatic product. In some embodiments the T cell antigen is a‘universal’ T cell antigen (i.e., one which can be used with anunrelated B cell antigen, including a carbohydrate, to stimulate T cellhelp). In some embodiments, the nanocarrier further comprises atargeting moiety. Again, the targeting moieties mentioned here (and asdescribed in more detail herein) is in addition to the plurality ofmoieties that are present on the immunofeature surface and that providetargeting of the nanocarriers to APCs.

In some embodiments, the nanocarrier comprises both a B cell antigen anda T cell antigen. In some embodiments, the B cell antigen and the T cellantigen are different antigens. In some embodiments, the B cell antigenand the T cell antigen are the same antigen. In some embodiments, the Bcell antigen is on the immunofeature surface of the nanocarrier, on asecond surface of the nanocarrier (e.g., covalently or non-covalentlyassociated) or is both on the surface of the nanocarrier (e.g.,covalently or non-covalently associated) and encapsulated within thenanocarrier (e.g., covalently or non-covalently associated), while the Tcell antigen is on the immunofeature surface of the nanocarrier, on thesecond surface of the nanocarrier (e.g., covalently or non-covalentlyassociated), is encapsulated within the nanocarrier (e.g., covalently ornon-covalently associated), or is both on the surface of the nanocarrier(e.g., covalently or non-covalently associated) and encapsulated withinthe nanocarrier (e.g., covalently or non-covalently associated).

In some embodiments, where a nanocarrier comprises both a B cell antigenand a T cell antigen, the nanocarrier further comprises animmunostimulatory agent. In some embodiments, the immunostimulatoryagent is on the surface of the nanocarrier and/or is encapsulated withinthe nanocarrier. In some embodiments, the immunostimulatory agent isassociated with the nanocarrier. In some embodiments, theimmunostimulatory agent is covalently associated with the nanocarrier.In some embodiments, the immunostimulatory agent is non-covalentlyassociated with the nanocarrier.

In some embodiments, where a nanocarrier comprises both a B cell antigenand a T cell antigen, the nanocarrier further comprises targeting agent.Again, the targeting agent mentioned here (and as described in moredetail herein) is in addition to the plurality of moieties that arepresent on the immunofeature surface and that provide targeting of thenanocarriers to APCs. In some embodiments, the targeting agent is on theimmunofeature surface of the nanocarrier, or on a second surface of thenanocarrier. In some embodiments, the targeting agent is associated withthe nanocarrier. In some embodiments, the targeting agent is covalentlyassociated with the nanocarrier. In some embodiments, the targetingagent is non-covalently associated with the nanocarrier.

In some embodiments, where a nanocarrier comprises both a B cell antigenand a T cell antigen, the nanocarrier further comprises animmunostimulatory agent and a targeting agent. In some embodiments, theimmunostimulatory agent is on the surface of the nanocarrier (e.g.,covalently or non-covalently associated) and/or is encapsulated withinthe nanocarrier (e.g., covalently or non-covalently associated), whilethe targeting agent is on the surface of the nanocarrier (e.g.,covalently or non-covalently associated).

In some embodiments, the nanocarrier comprises an immunostimulatoryagent. In some embodiments, the immunostimulatory agent is on thesurface of the nanocarrier. In some embodiments, the immunostimulatoryagent is encapsulated within the nanocarrier. In some embodiments, theimmunostimulatory agent is both on the surface of the nanocarrier andencapsulated within the nanocarrier. In some embodiments, theimmunostimulatory agent on the surface of the nanocarrier is differentfrom the immunostimulatory agent encapsulated within the nanocarrier. Insome embodiments, the immunostimulatory agent on the surface of andencapsulated within the nanocarrier is the same.

In some embodiments, the nanocarrier comprises more than one species ofimmunostimulatory agents, in which case the immunostimulatory agents aredifferent.

In some embodiments, the nanocarrier comprises an immunofeature surface,an immunostimulatory agent and an antigen. In some embodiments, theantigen is a B cell antigen or a T cell antigen. In some embodiments,the immunostimulatory agent is an immunosuppressant (suppresses animmune response). In some embodiments, the immunosuppressant iscyclosporin, a steroid, methotrexate or any agent that interferes with Tcell activation. In some embodiments, the immunostimulatory agentinduces regulatory T cells (e.g., TGF-β, rapamycin or retinoic acid). Insome embodiments, the immunosuppressant or agent that induces regulatoryT cells promotes the acquisition of tolerance to an antigen. Thenanocarrier, in some embodiments, further comprises a targeting agent.In some embodiments, the nanocarrier can be used to suppress the immunesystem and/or promote tolerance in a subject.

In some embodiments where the nanocarrier comprises an immunofeaturesurface, and an immunostimulatory agent, the nanocarrier furthercomprises a B cell antigen and/or a T cell antigen. In some embodiments,the B cell antigen is a poorly immunogenic antigen. In some embodiments,the B cell antigen is a small molecule. In some embodiments, the B cellantigen is a carbohydrate. In some embodiments, the B cell antigen is anaddictive substance. In some embodiments, the B cell antigen is a toxin.In some embodiments, the T cell antigen is a degenerative diseaseantigen, an infectious disease antigen, a cancer antigen, an atopicdisease antigen, an autoimmune disease antigen, an alloantigen, axenoantigen, an addictive substance, or a metabolic disease enzyme orenzymatic product. In some embodiments, the T cell antigen is anuniversal T cell antigen. In some embodiments, the nanocarrier furthercomprises a targeting agent.

The nanocarrier, in some embodiments, can be used to induce or enhancean immune response to a poorly immunogenic antigen (e.g., a smallmolecule or carbohydrate) in a subject. In some embodiments, thenanocarrier can be used to induce or enhance an immune response to anaddictive substance in a subject. In some embodiments, the nanocarriercan be used to induce or enhance an immune response to a toxin in asubject. The nanocarrier, in some embodiments, can be used to treat asubject that has or is susceptible to an addiction. The nanocarrier, insome embodiments, can be used to treat a subject that has been or willbe exposed to a toxin. In some embodiments, the nanocarrier can be usedto treat and/or prevent infectious disease, cancer, or autoimmunedisease (including rheumatoid arthritis). In other embodiments, thenanocarriers can be used for immune suppression in connection withtransplants to ameliorate transplant rejection.

In some embodiments, the nanocarrier further comprises a targetingmoiety. In some embodiments, the targeting moiety is on theimmunofeature surface, or on the second surface of the nanocarrier. Insome embodiments, the targeting moiety is associated with thenanocarrier. In some embodiments, the targeting moiety is covalentlyassociated with the nanocarrier. In some embodiments, the targetingmoiety is non-covalently associated with the nanocarrier.

In some aspects a composition comprising a nanocarrier comprising (a) aconjugate of a polymer and an immunofeature moiety (i.e., one of theplurality of moieties on an immunofeature surface), (b) a conjugate of apolymer and an antigen, (c) a conjugate of a polymer and animmunostimulatory agent, and/or (d) a conjugate of a polymer and atargeting moiety is provided. In some embodiments, the nanocarriercomprises a conjugate of a polymer and an antigen and a conjugate of apolymer and an immunostimulatory agent. In some embodiments, thenanocarrier comprises a conjugate of a polymer and an antigen and aconjugate of a polymer and a targeting moiety. In some embodiments, thenanocarrier comprises a conjugate of a polymer and an immunostimulatoryagent and a conjugate of a polymer and a targeting moiety. In someembodiments, the nanocarrier comprises a conjugate of a polymer and anantigen, a conjugate of a polymer and an immunostimulatory agent and aconjugate of a polymer and a targeting moiety. In some embodiments, theconjugate or conjugates is/are covalent conjugate/conjugates ornon-covalent conjugate/conjugates or any combination thereof. In someembodiments, the antigen is a B cell antigen. In some embodiments, thenanocarrier further comprises a conjugate of a polymer and a T cellantigen. In some embodiments, such a conjugate is a covalent ornon-covalent conjugate. In some embodiments, the antigen is a T cellantigen. In some embodiments, the nanocarrier further comprises aconjugate of a polymer and a B cell antigen. In some embodiments, such aconjugate is a covalent or non-covalent conjugate.

In some aspects, a composition comprising a nanocarrier comprising amolecule or molecules of the following formula X-L1-Y-L2-Z, wherein X isa biodegradable polymer, Y is a water soluble, non-adhesive polymer, Zis a targeting moiety, an immunomodulatory agent, an immunostimulatoryagent, or a pharmaceutical agent, and L1 and L2 are bonds or linkingmolecules, wherein either Y or Z, but not both Y and Z, can be absent,is provided. In some embodiments, the nanocarrier comprises an antigen,an immunostimulatory agent, or both. In some embodiments, thepharmaceutical agent is an antigen. In some embodiments, the antigen isa degenerative disease antigen, an infectious disease antigen, a cancerantigen, an atopic disease antigen, an autoimmune disease antigen, analloantigen, a xenoantigen, an addictive substance, or a metabolicdisease enzyme or enzymatic product. Z may be any antigen describedherein. In some embodiments, Z is a targeting moiety. In someembodiments, Z is a targeting moiety that binds a receptor expressed onthe surface of a cell. In some embodiments, Z is a targeting moiety thatbinds a soluble receptor. In some embodiments, the soluble receptor is acomplement protein or a pre-existing antibody. In some embodiments, thetargeting moiety is for delivery of the nanocarrier to antigenpresenting cells, T cells or B cells. In some embodiments, the antigenpresenting cells are dendritic cells (DCs), follicular dendritic cells(FDCs), or macrophages. In some embodiments, the macrophages aresubcapsular sinus macrophages (SCS-Mphs). In some embodiments, the Y isPEG or PEO. In some embodiments, Y is polyalkylene glycol orpolyalkylene oxide. In some embodiments, X is PLGA, PLA or PGA. In someembodiments, Z is absent.

The nanocarriers of the invention comprise a surface comprising animmunofeature surface. In some aspects, the composition comprises ananocarrier comprising a molecule or molecules of the following formula:X-L1-Y-L2-I, wherein X, L1, Y, and L2 are as described previously and Iis an immunofeature moiety (i.e., one of the plurality of moieties onthe immunofeature surface). As described herein, the immunofeaturesurface (via the plurality of moieties on the immunofeature surface)provides low affinity, high avidity binding to APCs.

In some aspects, a composition comprising a nanocarrier comprising animmunofeature surface is provided. In some aspects, a compositioncomprising a nanocarrier comprising an immunostimulatory agent isprovided. In some embodiments, the composition further comprises anantigen and/or a targeting moiety. In some embodiments, at least one ofthe antigen, targeting moiety, and immunostimulatory agent is conjugatedto a water soluble, non-adhesive polymer. In some embodiments, at leastone of the antigen, targeting moiety, and immunostimulatory agent isconjugated to a biodegradable polymer. In some embodiments, at least oneof the antigen, targeting moiety, and immunostimulatory agent isconjugated to a biocompatible polymer. In some embodiments, thebiocompatible polymer is a conjugate of a water soluble, non-adhesivepolymer conjugated to a biodegradable polymer. In some embodiments, theantigen is a B cell antigen. In some embodiments, the B cell antigen isnot a T cell antigen. In some embodiments, the nanocarrier furthercomprises a T cell antigen. In some embodiments, the antigen is a T cellantigen.

In some aspects, a composition comprising a nanocarrier comprising animmunofeature surface, a small molecule, an immunostimulatory agent, anda T cell antigen is provided. In some embodiments, the small molecule ison the immunofeature surface, a second surface of the nanocarrier or isboth on the surface of the nanocarrier and encapsulated within thenanocarrier. In some embodiments, the small molecule is an addictivesubstance. In some embodiments, the addictive substance is nicotine. Insome embodiments, the small molecule is a toxin. In some embodiments,the toxin is from a chemical weapon, an agent of biowarfare, or ahazardous environmental agent. In some embodiments, the small moleculeis conjugated to a polymer. In some embodiments, the polymer is a watersoluble, non-adhesive polymer, a biodegradable polymer, or abiocompatible polymer. In some embodiments, the polymer is abiocompatible polymer. In some embodiments, the immunostimulatory agentis on the surface of the nanocarrier or is both on the surface of thenanocarrier and encapsulated within the nanocarrier. In someembodiments, the immunostimulatory agent is conjugated to a polymer. Insome embodiments, the polymer is a water soluble, non-adhesive polymer,a biodegradable polymer, or a biocompatible polymer biodegradablepolymer. In some embodiments, the polymer is water soluble, non-adhesivepolymer or a biodegradable polymer. In some embodiments, the nanocarrierfurther comprises a targeting moiety. In some embodiments, the targetingmoiety is conjugated to a polymer. In some embodiments, the polymer is awater soluble, non-adhesive polymer, a biodegradable polymer, or abiocompatible polymer biodegradable polymer. In some embodiments, thepolymer is a biocompatible polymer. In some embodiments, the watersoluble, non-adhesive polymer is PEG or PEO. In some embodiments thewater soluble, non-adhesive polymer is polyalkylene glycol orpolyalkylene oxide. In some embodiments, the biodegradable polymer isPLGA, PLA, or PGA. In some embodiments, the biocompatible polymer is aconjugate of a water soluble, non-adhesive polymer and a biodegradablepolymer.

In some embodiments, a composition comprising a nanocarrier comprisingnicotine, an immunostimulatory agent, a T cell antigen, and a targetingmoiety is provided. In some embodiments, the immunostimulatory agent isa TLR 7/8 agonist. In some embodiments, the immunostimulatory agent isR848 (also referred to as CL097) or imiquimod. In some embodiments, thenicotine is on the surface of the nanocarrier or is both on the surfaceof the nanocarrier and encapsulated within the nanocarrier. In someembodiments, the nicotine is conjugated to a polymer, preferablycovalently conjugated. In some embodiments, the polymer is a watersoluble, non-adhesive polymer, a biodegradable polymer, or abiocompatible polymer. In some embodiments, the nicotine is conjugatedto a biocompatible polymer. In some embodiments, the immunostimulatoryagent is on the surface of the nanocarrier, is encapsulated within thenanocarrier, or is both on the surface of the nanocarrier andencapsulated within the nanocarrier. In some embodiments, theimmunostimulatory agent is conjugated to a polymer. In some embodiments,the polymer is a water soluble, non-adhesive polymer, a biodegradablepolymer, or a biocompatible polymer biodegradable polymer. In someembodiments, immunostimulatory agent is conjugated to a biodegradablepolymer. In some embodiments, the targeting moiety is conjugated to apolymer. In some embodiments, the polymer is a water soluble,non-adhesive polymer, a biodegradable polymer, or a biocompatiblepolymer biodegradable polymer. In some embodiments, targeting moiety isconjugated to a biocompatible polymer. In some embodiments, the watersoluble, non-adhesive polymer is PEG or PEO. In some embodiments thewater soluble, non-adhesive polymer is polyalkylene glycol orpolyalkylene oxide. In some embodiments, the biodegradable polymer isPLGA, PLA, or PGA. In some embodiments, the biocompatible polymer is aconjugate of a water soluble, non-adhesive polymer and a biodegradablepolymer.

In some aspects, a composition comprising a nanocarrier comprising apoorly immunogenic antigen, an immunostimulatory agent, and a T cellantigen is provided. In some embodiments, the poorly immunogenic antigenis on the surface of the nanocarrier or is both on the surface of thenanocarrier and encapsulated within the nanocarrier. In someembodiments, the poorly immunogenic antigen is a small molecule or acarbohydrate. In some embodiments, the poorly immunogenic antigen is anaddictive substance. In some embodiments, the poorly immunogenic antigenis a toxin. In some embodiments, the poorly immunogenic antigen iscovalently conjugated to a polymer. In some embodiments, the polymer isa water soluble, non-adhesive polymer, a biodegradable polymer, or abiocompatible polymer biodegradable polymer. In some embodiments, theimmunostimulatory agent is on the surface of the nanocarrier or is bothon the surface of the nanocarrier and encapsulated within thenanocarrier. In some embodiments, the immunostimulatory agent iscovalently conjugated to a polymer. In some embodiments, the polymer isa water soluble, non-adhesive polymer, a biodegradable polymer, or abiocompatible polymer biodegradable polymer. In some embodiments, thenanocarrier further comprises a targeting moiety. In some embodiments,the targeting moiety is covalently conjugated to a polymer. In someembodiments, the polymer is a water soluble, non-adhesive polymer, abiodegradable polymer, or a biocompatible polymer biodegradable polymer.

In some aspects, a composition comprising a nanocarrier that targets aspecific cell, tissue or organ and modulates an immune responsecomprising a B cell antigen on its surface at a density that activates Bcells and a immunostimulatory agent is provided. In some embodiments,the nanocarrier further comprises a targeting moiety. In someembodiments, the composition is a pharmaceutical composition and furthercomprises a pharmaceutically acceptable carrier. In some embodiments,the pharmaceutical composition is a vaccine composition.

In some aspects, a composition, such as a pharmaceutical composition,comprising an antigen presenting cell-targeting moiety and a nanocarrieris provided. In some embodiments, the antigen presenting cell-targetingmoiety and nanocarrier are conjugated. In some embodiments, theconjugate is a covalent conjugate. In some embodiments, the conjugate isa non-covalent conjugate.

In some aspects, a composition, such as a pharmaceutical composition,comprising an immunostimulatory agent and a nanocarrier is provided. Insome embodiments, the immunostimulatory agent and nanocarrier areconjugated. In some embodiments, the conjugate is a covalent conjugate.In some embodiments, the conjugate is a non-covalent conjugate.

In some aspects, a composition comprising a molecule with the formulaX-L1-Y-L2-Z, wherein X is a biodegradable polymer, Y is a water soluble,non-adhesive polymer, Z is a targeting moiety, an immunostimulatoryagent, or a pharmaceutical agent, and L1 and L2 are bonds or linkingmolecules, wherein either Y or Z, but not both Y and Z, can be absent isprovided.

In some aspects, a composition comprising a molecule with the formulaX-L1-Y-L2-I, wherein I is an immunofeature moiety, X is a biodegradablepolymer, Y is a water soluble, non-adhesive polymer, and L1 and L2 arebonds or linking molecules, wherein either Y or I, but not both Y and I,can be absent is provided.

In some aspects, a composition comprising a molecule with the formulaT-L1-X-L2-Y-L3-Z, where T is a T cell antigen, X is a biodegradablepolymer, Y is a water soluble, non-adhesive polymer, Z is an Z is atargeting moiety, an immunostimulatory agent, or a pharmaceutical agent,wherein L1, L2, and L3 are bonds or linking molecules, and wherein anyone or two of T, Y, and Z, but not all three of T, Y, and Z, can beabsent is provided. In some embodiments, the pharmaceutical agent is anantigen. In some embodiments, the antigen is a B cell antigen or a Tcell antigen.

In some aspects, a composition comprising a molecule with the formulaT-L1-X-L2-Y-L3-I, where I is an immunofeature moiety, T is a T cellantigen, X is a biodegradable polymer, Y is a water soluble,non-adhesive polymer, Z is an Z is a targeting moiety, animmunostimulatory agent, or a pharmaceutical agent, wherein L1, L2, andL3 are bonds or linking molecules, and wherein any one or two of T, Y,and Z, but not all three of T, Y, and Z, can be absent is provided. Insome embodiments, the pharmaceutical agent is an antigen. In someembodiments, the antigen is a B cell antigen or a T cell antigen.

In some embodiments, Z is a degenerative disease antigen, an infectiousdisease antigen, a cancer antigen, an atopic disease antigen, anautoimmune disease antigen, an alloantigen, a xenoantigen, a hapten, anaddictive substance, or a metabolic disease enzyme or enzymatic product.In some embodiments, Z is any of the B cell antigens described herein.In some embodiments, Z is any of the T cell antigens provided herein.

In some embodiments, Z is a targeting moiety that binds a receptorexpressed on the surface of a cell. In some embodiments, Z is atargeting moiety that binds a soluble receptor. In some embodiments, thesoluble receptor is complement or a pre-existing antibody. In someembodiments, the targeting moiety is for targeting antigen presentingcells, T cells or B cells.

In some embodiments, Y is PEG or PEO. In some embodiments, Y ispolyalkylene glycol or polyalkylene oxide.

In some embodiments, X is PLGA, PGA, or PLA.

In some embodiments, Z is absent. In some embodiments, Y is absent.

In some aspects, a pharmaceutical composition comprising a conjugate ofa immunostimulatory agent and a polymer is provided. In someembodiments, the conjugate is a covalent conjugate. In some embodiments,the conjugate is a non-covalent conjugate. In some embodiments, thepolymer is a water soluble, non-adhesive polymer, a biodegradablepolymer, or a biocompatible polymer. In some embodiments, the polymer isa biocompatible polymer. In some embodiments, the biocompatible polymeris a biodegradable polymer or a water soluble, non-adhesive polymer. Insome embodiments, the biocompatible polymer is a conjugate of a watersoluble, non-adhesive polymer and a biodegradable polymer. In someembodiments, the polymer is synthetic. In some embodiments, thepharmaceutical composition comprises one or more nanocarriers whereinthe conjugate is a component of the one or more nanocarriers. In someembodiments, the composition further comprises an antigen. In someembodiments, the pharmaceutical composition does not comprise anantigen. In some embodiments, the composition further comprises atargeting agent.

In some aspects, a vaccine composition comprising a conjugate of animmunostimulatory agent and a polymer is provided. In some embodiments,the conjugate is a covalent conjugate. In some embodiments, theconjugate is a non-covalent conjugate. In some embodiments, the polymeris a water soluble, non-adhesive polymer, a biodegradable polymer, or abiocompatible polymer. In some embodiments, the water soluble,non-adhesive polymer is polyethylene glycol. In some embodiments, thepolymer is a biocompatible polymer. In some embodiments, thebiocompatible polymer is a biodegradable polymer or a water soluble,non-adhesive polymer. In some embodiments, the biocompatible polymer isa conjugate of a water soluble, non-adhesive polymer and a biodegradablepolymer. In some embodiments, the polymer is synthetic. In someembodiments, the pharmaceutical composition comprises one or morenanocarriers wherein the conjugate is a component of the one or morenanocarriers. In some embodiments, the composition further comprises anantigen. In some embodiments, the pharmaceutical composition does notcomprise an antigen. In some embodiments, the composition furthercomprises a targeting agent.

In some embodiments, the B cell antigen is a protein or peptide. In someembodiments, the B cell antigen is a non-protein antigen (i.e., not aprotein or peptide). In some embodiments, the protein or peptide is froman infectious agent. In some embodiments, the infectious agent is abacterium, fungus, virus, protozoan, or parasite. In some embodiments,the virus is a pox virus, smallpox virus, ebola virus, marburg virus,dengue fever virus, influenza virus, parainfluenza virus, respiratorysyncytial virus, rubeola virus, human immunodeficiency virus, humanpapillomavirus, varicella-zoster virus, herpes simplex virus,cytomegalovirus, Epstein-Barr virus, JC virus, rhabdovirus, rotavirus,rhinovirus, adenovirus, papillomavirus, parvovirus, picornavirus,poliovirus, virus that causes mumps, virus that causes rabies, reovirus,rubella virus, togavirus, orthomyxovirus, retrovirus, hepadnavirus,coxsackievirus, equine encephalitis virus, Japanese encephalitis virus,yellow fever virus, Rift Valley fever virus, hepatitis A virus,hepatitis B virus, hepatitis C virus, hepatitis D virus, or hepatitis Evirus.

In some embodiments, the B cell antigen is a small molecule. In someembodiments, the small molecule is an abused substance, an addictivesubstance, or a toxin.

In some embodiments, the B cell antigen is an addictive substance. Insome embodiments, the addictive substance is nicotine, a narcotic, ahallucinogen, a stimulant, a cough suppressant, a tranquilizer, or asedative. In some embodiments, the B cell antigen is an opiod orbenzodiazepine.

In some embodiments, the B cell antigen is a toxin. In some embodiments,the toxin is from a chemical weapon. In some embodiments, the toxin froma chemical weapon is botulinum toxin or phosphene. Toxins from achemical weapon also include, but are not limited to, O-Alkyl (<C10,incl. cycloalkyl) alkyl (Me, Et, n-Pr or i-Pr)-phosphonofluoridates(e.g. Sarin: O-Isopropyl methylphosphonofluoridate, Soman: O-Pinacolylmethylphosphonofluoridate), O-Alkyl (<C10, incl. cycloalkyl) N,N-dialkyl(Me, Et, n-Pr or i-Pr) phosphoramidocyanidates (e.g. Tabun: O-EthylN,N-dimethylphosphoramidocyanidate), O-Alkyl (H or <C10, incl.cycloalkyl) S-2-dialkyl (Me, Et, n-Pr or i-Pr)-aminoethyl alkyl (Me, Et,n-Pr or i-Pr) phosphonothiolates and corresponding alkylated orprotonated salts (e.g. VX: O-Ethyl S-2-diisopropylaminoethylmethylphosphonothiolate), Sulfur mustards:2-Chloroethylchloromethylsulfide, Mustard gas:Bis(2-chloroethyl)sulfide, Bis(2-chloroethylthio)methane, Sesquimustard:1,2-Bis(2-chloroethylthio)ethane, 1,3-Bis(2-chloroethylthio)-n-propane,1,4-Bis(2-chloroethylthio)-n-butane,1,5-Bis(2-chloroethylthio)-n-pentane, Bis(2-chloroethylthiomethyl)ether,O-Mustard: Bis(2-chloroethylthioethyl)ether, Lewisites: Lewisite 1:2-Chlorovinyldichloroarsine, Lewisite 2: Bis(2-chlorovinyl)chloroarsine,Lewisite 3: Tris(2-chlorovinyl)arsine, Nitrogen mustards: HN1:Bis(2-chloroethyl)ethylamine, HN2: Bis(2-chloroethyl)methylamine, HN3:Tris(2-chloroethyl)amine, Saxitoxin, Ricin, Amiton: O,O-DiethylS-(2-(diethylamino)ethyl)phosphorothiolate and corresponding alkylatedor protonated salts, PFIB:1,1,3,3,3-Pentafluoro-2-(trifluoromethyl)-1-propene, 3-Quinuclidinylbenzilate (BZ), Phosgene: Carbonyl dichloride, Cyanogen chloride,Hydrogen cyanide and Chloropicrin: Trichloronitromethane. In someembodiments, the toxin for inclusion in a nanocarrier is a completemolecule of any of the foregoing or a portion thereof.

In some embodiments, the B cell antigen is a biohazard or hazardousenvironmental agent. In some embodiments, the hazardous environmentalagent is arsenic, lead, mercury, vinyl chloride, polychlorinatedbiphenyls, benzene, polycyclic aromatic hydrocarbons, cadmium,benzo(a)pyrene, benzo(b)fluoranthene, chloroform,dichlor-diphenyl-trichlorethylene (DDT), P,P′-, aroclor 1254, aroclor1260, dibenzo(a,h)anthracene, trichloroethylene, dieldrin, chromiumhexavalent, or p,p′-dichlorodiphenyldichloroethene (DDE, P,P′).

In some embodiments, the B cell antigen is a carbohydrate. In someembodiments, the carbohydrate is from an infectious agent. In someembodiments, the infectious agent is a bacterium, fungus, virus,protozoan, or parasite. In some embodiments, the bacterium is aPseudomonas, Pneumococcus, E. coli, Staphylococcus, Streptococcus,Treponema, Borrelia, Chlamydia, Haemophilus, Clostridium, Salmonella,Legionella, Vibrio or Enterococci bacterium or a Mycobacterium. In someembodiments, the virus is a pox virus, smallpox virus, ebola virus,marburg virus, dengue fever virus, influenza virus, parainfluenza virus,respiratory syncytial virus, rubeola virus, human immunodeficiencyvirus, human papillomavirus, varicella-zoster virus, herpes simplexvirus, cytomegalovirus, Epstein-Barr virus, JC virus, rhabdovirus,rotavirus, rhinovirus, adenovirus, papillomavirus, parvovirus,picornavirus, poliovirus, virus that causes mumps, virus that causesrabies, reovirus, rubella virus, togavirus, orthomyxovirus, retrovirus,hepadnavirus, coxsackievirus, equine encephalitis virus, Japaneseencephalitis virus, yellow fever virus, Rift Valley fever virus,hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis Dvirus, or hepatitis E virus.

In some embodiments, the B cell antigen is a self antigen. In someembodiments, the self antigen is a protein or peptide, lipoprotein,lipid, carbohydrate, or a nucleic acid. In some embodiments, the selfantigen is an enzyme, a structural protein, a secreted protein, a cellsurface receptor, or a cytokine. In some embodiments, the cytokine isTNF, IL-1, or IL-6. In some embodiments, the self antigen is cholesterylester transfer protein (CETP), the Aβ protein associated withAlzheimer's, a proteolytic enzyme that processes the pathological formof the Aβ protein, LDL associated with atherosclerosis, or a coreceptorfor HIV-1. In some embodiments, the proteolytic enzyme that processesthe pathological form of the Aβ protein is beta-secretase. In someembodiments, the LDL associated with atherosclerosis is oxidized orminimally modified. In some embodiments, the coreceptor for HIV-1 isCCR5. In some embodiments, the self antigen is an autoimmune diseaseantigen.

In some embodiments, the B cell antigen is a degenerative diseaseantigen, an infectious disease antigen, a cancer antigen, an atopicdisease antigen, an autoimmune disease antigen, or a metabolic diseaseenzyme or enzymatic product thereof.

In some embodiments, the antigen is a cancer antigen. Is someembodiments, the cancer antigen is Melan-A/MART-1, Dipeptidyl peptidaseIV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b,Colorectal associated antigen (CRC)-C017-1A/GA733, CarcinoembryonicAntigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1,Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-1,PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T-cellreceptor/CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-A1,MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9,MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3),MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-05),GAGE-family of tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4,GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V,MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1,α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn,gp100^(Pmel117), PRAME, NY-ESO-1, brain glycogen phosphorylase, SSX-1,SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7, cdc27, adenomatouspolyposis coli protein (APC), fodrin, P1A, Connexin 37, Ig-idiotype,p15, gp75, GM2 and GD2 gangliosides, viral products such as humanpapilloma virus proteins, Smad family of tumor antigens, 1mp-1,EBV-encoded nuclear antigen (EBNA)-1, or c-erbB-2.

In some embodiments, the infectious disease antigen is a viral antigen.In some embodiments, the viral antigen is an antigen from a pox virus,smallpox virus, ebola virus, marburg virus, dengue fever virus,influenza virus, parainfluenza virus, respiratory syncytial virus,rubeola virus, human immunodeficiency virus, human papillomavirus,varicella-zoster virus, herpes simplex virus, cytomegalovirus,Epstein-Barr virus, JC virus, rhabdovirus, rotavirus, rhinovirus,adenovirus, papillomavirus, parvovirus, picornavirus, poliovirus, virusthat causes mumps, virus that causes rabies, reovirus, rubella virus,togavirus, orthomyxovirus, retrovirus, hepadnavirus, coxsackievirus,equine encephalitis virus, Japanese encephalitis virus, yellow fevervirus, Rift Valley fever virus, hepatitis A virus, hepatitis B virus,hepatitis C virus, hepatitis D virus, or hepatitis E virus.

In some embodiments, the B cell antigen is a poorly immunogenic antigen.In some embodiments, the poorly immunogenic antigen is a non-proteinantigen. In some embodiments, the poorly immunogenic antigen is acarbohydrate or small molecule. In some embodiments, the poorlyimmunogenic antigen is an abused substance, addictive substance, ortoxin. In some embodiments, the toxin is from a chemical weapon. In someembodiments, the poorly immunogenic antigen is a hazardous environmentalagent. In some embodiments, the poorly immunogenic antigen is a selfantigen.

In general, the T cell antigen is a protein or peptide. In someembodiments, the T cell antigen is a degenerative disease antigen, aninfectious disease antigen, a cancer antigen, an atopic disease antigen,an autoimmune disease antigen, an alloantigen, a xenoantigen, a contactsensitizer, a hapten, or a metabolic disease enzyme or enzymaticproduct.

In some embodiments, the T cell antigen is from an infectious agent. Insome embodiments, the infectious agent is a bacterium, fungus, virus,protozoan, or parasite. In some embodiments, the infectious diseaseantigen is a viral antigen. In some embodiments, the viral antigen is anantigen from a pox virus, smallpox virus, ebola virus, marburg virus,dengue fever virus, influenza virus, parainfluenza virus, respiratorysyncytial virus, rubeola virus, human immunodeficiency virus, humanpapillomavirus, varicella-zoster virus, herpes simplex virus,cytomegalovirus, Epstein-Barr virus, JC virus, rhabdovirus, rotavirus,rhinovirus, adenovirus, papillomavirus, parvovirus, picornavirus,poliovirus, virus that causes mumps, virus that causes rabies, reovirus,rubella virus, togavirus, orthomyxovirus, retrovirus, hepadnavirus,coxsackievirus, equine encephalitis virus, Japanese encephalitis virus,yellow fever virus, Rift Valley fever virus, hepatitis A virus,hepatitis B virus, hepatitis C virus, hepatitis D virus, or hepatitis Evirus.

In some embodiments, T cell antigen is a universal T cell antigen. Insome embodiments, the universal T cell antigen is one or more peptidesderived from tetanus toxoid, Epstein-Barr virus, or influenza virus.

In some embodiments, immunostimulatory agents are interleukins,interferon, cytokines, etc. In some embodiments, the immunostimulatoryagent is a toll-like receptor (TLR) agonist, cytokine receptor agonist,CD40 agonist, Fc receptor agonist, CpG-containing immunostimulatorynucleic acid, complement receptor agonist, or an adjuvant. In someembodiments, the TLR agonist is a TLR-1, TLR-2, TLR-3, TLR-4, TLR-5,TLR-6, TLR-7, TLR-8, TLR-9, or TLR-10 agonist. In some embodiments, theFc receptor agonist is a Fc-gamma receptor agonist. In some embodiments,the complement receptor agonist binds to CD21 or CD35. In someembodiments, the complement receptor agonist induces endogenouscomplement opsonization of the nanocarrier. In some embodiments, thecytokine receptor agonist is a cytokine. In some embodiments, thecytokine receptor agonist is a small molecule, antibody, fusion protein,or aptamer. In some embodiments, the immunostimulatory agent is anadjuvant. In some embodiments, the adjuvant induces cytokinebiosynthesis. In some embodiments, the adjuvant is alum, MF59, R848,cholera toxin, squalene, phosphate adjuvants, or tetrachlorodecaoxide.In some embodiments, the adjuvant is monophosphoryl lipid A (MPL,SmithKline Beecham); saponins including QS21 (SmithKline Beecham);immunostimulatory oligonucleotides (e.g., CpG immunostimulatoryoligonucleotides first described by Kreig et al., Nature 374:546-9,1995); incomplete Freund's adjuvant; complete Freund's adjuvant;montanide; vitamin E and various water-in-oil emulsions prepared frombiodegradable oils such as squalene and/or tocopherol, Quil A, RibiDetox, CRL-1005, or L-121.

In specific embodiments, an immunostimulatory agent may be a natural orsynthetic agonist for a Toll-like receptor (TLR). In specificembodiments, an immunostimulatory agent may be a ligand for toll-likereceptor (TLR)-7, such as CpGs, which induce type I interferonproduction; an agonist for the DC surface molecule CD40; an agent thatpromotes DC maturation; a TLR-4 agonist; a cytokine; proinflammatorystimuli released from necrotic cells (e.g. urate crystals); activatedcomponents of the complement cascade (e.g. CD21, CD35, etc.); and soforth.

In some embodiments, the targeting moiety binds a receptor expressed onthe surface of a cell. In some embodiments, the targeting moiety binds asoluble receptor. In some embodiments, the soluble receptor is acomplement protein or a pre-existing antibody. In some embodiments, thetargeting moiety is for delivery of the nanocarrier to antigenpresenting cells, T cells, or B cells. In some embodiments, the antigenpresenting cells are macrophages. In some embodiments, the macrophagesare subcapsular sinus macrophages. In some embodiments, the antigenpresenting cells are dendritic cells. In some embodiments, the antigenpresenting cells are follicular dendritic cells.

In some embodiments, the targeting moiety is a molecule that binds toCD11b, CD169, mannose receptor, DEC-205, CD11c, CD21/CD35, CX3CR1, or aFc receptor. In some embodiments, the targeting moiety is a moleculethat binds to CD169, CX3CR1, or a Fc receptor. In some embodiments, themolecule that binds to CD169 is an anti-CD169 antibody. In someembodiments, the molecule that binds CX3CR1 is CX3CL1 (fractalkine). Insome embodiments, the targeting moiety comprises the Fc portion of animmunoglobulin. In some embodiments, the targeting moiety comprises theFc portion of an IgG. In some embodiments, the Fc portion of animmunoglobulin is a human Fc portion of an immunoglobulin. In someembodiments, the Fc portion of an IgG is a human Fc portion of an IgG.In some embodiments, the targeting moiety is the soluble receptor, CRFc.In some embodiments, CRFc can be used to target macrophages in thesubcapsular sinus but not macrophages of the medulla. In someembodiments, the targeting moiety is one or more amine moieties.

In some aspects, the compositions provided herein are immunogenic.

In some aspects, a method comprising administering any of thecompositions provided herein to a subject in an amount effective tomodulate an immune response is provided. In some embodiments, thecomposition is in an amount effective to induce or enhance an immuneresponse. In some embodiments, the composition is in an amount effectiveto suppress an immune response. In some embodiments, the composition isin an amount effective to direct or redirect an immune response. In someembodiments, the method is for prophylaxis and/or treatment of theconditions identified herein.

In some embodiments, where the method is to induce or enhance an immuneresponse, the subject has or is susceptible to having cancer, aninfectious disease, a non-autoimmune metabolic or degenerative disease,an atopic disease, or an addiction. In some embodiments, the subject hasbeen exposed to or may be exposed to a toxin. In some embodiments, thesubject has been exposed to or may be exposed to a toxin from a chemicalweapon. In some embodiments, the subject has been exposed to or may beexposed to a toxin from a hazardous environmental substance. In someembodiments, the nanocarrier comprises a B-cell antigen, animmunostimulatory agent, and a T cell antigen, such as an universal Tcell antigen. In some embodiments, the nanocarrier further comprises atargeting moiety.

In some embodiments, where the method is for treating or preventing anaddiction (or for treating a subject exposed to or who may be exposed toa toxin), the nanocarrier comprises the addictive substance or toxin, anadjuvant, and a T cell. In some embodiments, the method raises hightiter antibodies that bind and neutralize the offending agent before itreaches its effector site (e.g., the brain). In some embodiments, theaddictive substance or toxin is at a high density on the surface of thenanocarrier.

In some embodiments, the infectious disease is a chronic viralinfection. In some embodiments, the chronic viral infection is HIV, HPV,HBV, or HCV infection. In some embodiments, the infectious disease is oris caused by a bacterial infection. In some embodiments, the subject hasor is susceptible to having a Pseudomonas infection, a Pneumococcusinfection, tuberculosis, malaria, leishmaniasis, H. pylori, aStaphylococcus infection, or a Salmonella infection. In someembodiments, the infectious disease is or is caused by a fungalinfection. In some embodiments, the infectious disease is or is causedby a parasitic infection. In some embodiments, the infectious disease isor is caused by a protozoan infection. In some embodiments, the subjecthas or is susceptible to having influenza.

In some embodiments, the autoimmune disease is disease is lupus,multiple sclerosis, rheumatoid arthritis, diabetes mellitus type I,inflammatory bowel disease, thyroiditis, or celiac disease. In someembodiments, the subject has had or will have a transplant, and themethod can be to prevent or ameliorate transplant rejection. In someembodiments, the nanocarrier comprises an antigen and an immunesuppressant or an agent that induces regulatory T cells. In someembodiments, the nanocarrier further comprises a targeting moiety.Generally, where the method is one to suppress an immune response, theantigen is provided in the absence of an adjuvant.

In some aspects, vaccine nanocarriers for delivery of immunomodulatoryagents to the cells of the immune system are provided. In someembodiments, vaccine nanocarriers comprise at least one immunomodulatoryagent that is capable of inducing an immune response in B cells and/orin T cells. In certain embodiments, immunomodulatory agents presented onnanocarrier surfaces stimulate B cells, and immunomodulatory agentsencapsulated within the nanocarriers are processed and presented to Tcells. In some embodiments, vaccine nanocarriers comprise at least onetargeting moiety that is useful for selective delivery of the vaccinenanocarrier to specific antigen-presenting cells (APCs).

In some embodiments, an immunomodulatory agent may comprise isolatedand/or recombinant proteins or peptides, carbohydrates, glycoproteins,glycopeptides, proteoglycans, inactivated organisms and viruses, deadorganisms and virus, genetically altered organisms or viruses, and cellextracts. In some embodiments, an immunomodulatory agent may comprisenucleic acids, carbohydrates, lipids, and/or small molecules. In someembodiments, an immunomodulatory agent is one that elicits an immuneresponse. In some embodiments, an immunomodulatory agent is an antigen.In some embodiments, an immunomodulatory agent is used for vaccines.

In some embodiments, an immunomodulatory agent is any protein and/orother antigen derived from a pathogen. The pathogen may be a virus,bacterium, fungus, protozoan, parasite, etc. In some embodiments, animmunomodulatory agent may be in the form of whole killed organisms,peptides, proteins, glycoproteins, glycopeptides, proteoglycans,carbohydrates, or combinations thereof.

In some embodiments, all of the immunomodulatory agents of a vaccinenanocarrier are identical to one another. In some embodiments, all ofthe immunomodulatory agents of a vaccine nanocarrier are different. Insome embodiments, a vaccine nanocarrier comprises exactly one distincttype (i.e., species) of immunomodulatory agent. For example, when theimmunomodulatory agent is an antigen, all of the antigens that are inthe vaccine nanocarrier are the same. In some embodiments, a vaccinenanocarrier comprises exactly two distinct types of immunomodulatoryagents. In some embodiments, a vaccine nanocarrier comprises greaterthan two distinct types of immunomodulatory agents.

In some embodiments, a vaccine nanocarrier comprises a single type ofimmunomodulatory agent that stimulates an immune response in B cells. Insome embodiments, a vaccine nanocarrier comprises a single type ofimmunomodulatory agent that stimulates an immune response in T cells. Insome embodiments, a vaccine nanocarrier comprises two types ofimmunomodulatory agents, wherein the first immunomodulatory agentstimulates B cells, and the second immunomodulatory agent stimulates Tcells. In certain embodiments, any of the aforementioned agents couldstimulate both B cells and T cells, but this is not necessarily so. Incertain embodiments, the aforementioned immunomodulatory agentsstimulates only B cells or T cells, respectively. In some embodiments, avaccine nanocarrier comprises greater than two types of immunomodulatoryagents, wherein one or more types of immunomodulatory agents stimulate Bcells, and one or more types of immunomodulatory agents stimulate Tcells.

In some embodiments, a vaccine nanocarrier includes a lipid membrane(e.g. lipid bilayer, lipid monolayer, etc.). At least oneimmunomodulatory agent may be associated with the lipid membrane. Insome embodiments, at least one immunomodulatory agent is embedded withinthe lipid membrane, embedded within the lumen of a lipid bilayer,associated with the interior surface of the lipid membrane, and/orencapsulated with the lipid membrane of a vaccine nanocarrier.

In some embodiments, a vaccine nanocarrier includes a polymer (e.g. apolymeric core). The immunomodulatory agent may be associated with thepolymer, and in some embodiments, at least one type of immunomodulatoryagent is associated with the polymer. In some embodiments, theimmunomodulatory agent is embedded within the polymer, associated withthe interior surface of the polymer, and/or encapsulated within thepolymer of a vaccine nanocarrier, and, in some embodiments, at least onetype of immunomodulatory agent is embedded within the polymer,associated with the interior surface of the polymer, and/or encapsulatedwithin the polymer of a vaccine nanocarrier.

In some embodiments, inventive vaccine nanocarriers comprise less thanless than 90% by weight, less than 75% by weight, less than 50% byweight, less than 40% by weight, less than 30% by weight, less than 20%by weight, less than 15% by weight, less than 10% by weight, less than5% by weight, less than 1% by weight, or less than 0.5% by weight of theimmunomodulatory agent.

In some embodiments, vaccine nanocarriers are associated with at leastone targeting moiety in addition to the plurality of moieties associatedwith the immunofeature surface (i.e., the moieties that providetargeting of the nanocarriers to APCs). The additional targetingmoieties are distinct from the plurality of moieties present on theimmunofeature surface in that the additional targeting moietiestypically provide high affinity binding to a receptor (and, therefore,may be alternatively referred to herein as “high affinity targetingmoieties”). In some embodiments, a targeting moiety may be a nucleicacid, polypeptide, peptide, glycoprotein, glycopeptide, proteoglycan,carbohydrate, lipid, small molecule, etc. For example, a targetingmoiety can be a nucleic acid targeting moiety (e.g. an aptamer,Spiegelmer®, etc.) that binds to a cell type specific marker. In someembodiments, a targeting moiety may be a naturally occurring orsynthetic ligand for a cell surface protein, e.g., DEC-205, CD169,CD11b, etc. Examples of targeting moieties also include those providedelsewhere herein, such as those described above.

In accordance with the present invention, a targeting moiety recognizesone or more “receptors,” “targets,” or “markers” associated with aparticular organ, tissue, cell, and/or subcellular locale. In someembodiments, a target may be a marker that is exclusively or primarilyassociated with one or a few cell types, with one or a few diseases,and/or with one or a few developmental stages. Examples of cells thatare targeted include antigen presenting cells (APCs), such as dendriticcells, follicular dendritic cells, and macrophages. One example of amacrophage is a subcapsular sinus macrophage. Other cells that aretargeted include T cells and B cells. In some embodiments, a target cancomprise a protein, a carbohydrate, a lipid, and/or a nucleic acid. Insome embodiments, a target is a tumor marker. In some embodiments, atarget is an APC marker. In certain embodiments, a target is a T cellmarker. In some embodiments, the targeting moieties target secondarylymphoid tissues or organs. Secondary lymphoid tissues or organs includelymph nodes, the spleen, Peyer's patches, the appendix, or tonsils.

In certain embodiments, a target is a dendritic cell marker. In someembodiments, DC markers include DC-205, CD11c, class II MHC, CD80, CD86,DC-SIGN, CD11b, BDCA-1, BDCA-2, BDCA-4, Siglec-H, CX3CR1, and/orLangerin. Examples of such markers are provided elsewhere herein.

In certain embodiments, a target is a subcapsular sinus macrophagemarker. In some embodiments, SCS-Mph markers include CD169 (i.e.sialoadhesin), CD11b (i.e. CD11b/CD18, Mac-1, CR3 or αMβ2 integrin), Fcreceptor, and/or the mannose receptor (i.e. a multi-valent lectin),proteins which are all prominently expressed on SCS-Mph. Examples ofsuch markers are provided elsewhere herein.

In certain embodiments, a target is a B cell marker. In someembodiments, B cell markers may include complement receptors, CR1 (i.e.CD35) or CR2 (i.e. CD21), proteins which are expressed on B cells. Insome embodiments, B cell targeting can be accomplished by B cell markerssuch as CD19, CD20, and/or CD22. In some embodiments, B cell targetingcan be accomplished by B cell markers such as CD40, CD52, CD80, CXCR5,VLA-4, class II MHC, surface IgM or IgD, APRL, and/or BAFF-R. Examplesof such markers are provided elsewhere herein.

In certain embodiments, a target is a FDC marker. In some embodiments,FDC markers include complement receptors, CR1 (i.e. CD35) or CR2 (i.e.CD21), proteins which are expressed on FDCs. Examples of such markersare provided elsewhere herein.

In some embodiments, a vaccine nanocarrier comprises a single type oftargeting moiety that directs delivery of the vaccine nanocarrier to asingle cell type (e.g. delivery to SCS-Mph only). In some embodiments, avaccine nanocarrier comprises a single type of targeting moiety thatdirects delivery of the vaccine nanocarrier to multiple cell types (e.g.delivery to both SCS-Mphs and FDCs, or to both SCS-Mphs and DCs). Insome embodiments, a vaccine nanocarrier comprises two types of targetingmoieties, wherein the first type of targeting moiety directs delivery ofthe vaccine nanocarrier to one cell type, and the second type oftargeting moiety directs delivery of the vaccine nanocarrier to a secondcell type. For example, in some embodiments, the first type of targetingmoiety directs delivery to SCS-Mphs, and the second type of targetingmoiety directs delivery to DCs. As another example, the first type oftargeting moiety directs delivery to SCS-Mphs, and the second type oftargeting moiety directs delivery to FDCs.

In some embodiments, inventive vaccine nanocarriers comprise less than50% by weight, less than 40% by weight, less than 30% by weight, lessthan 20% by weight, less than 15% by weight, less than 10% by weight,less than 5% by weight, less than 1% by weight, or less than 0.5% byweight of the targeting moiety.

In some embodiments, vaccine nanocarriers may transport one or moretypes of immunostimulatory agents which can help stimulate immuneresponses. In some embodiments, immunostimulatory agents boost immuneresponses by activating APCs to enhance their immunostimulatorycapacity. In some embodiments, immunostimulatory agents boost immuneresponses by amplifying lymphocyte responses to specific antigens. Insome embodiments, immunostimulatory agents boost immune responses byinducing the local release of mediators, such as cytokines from avariety of cell types.

In some embodiments, a vaccine nanocarrier comprises a single type ofimmunostimulatory agent that stimulates both B cells and T cells. Insome embodiments, a vaccine nanocarrier comprises two types ofimmunostimulatory agents, wherein the first type of immunostimulatoryagent stimulates B cells, and the second type of immunostimulatory agentstimulates T cells. In some embodiments, a vaccine nanocarrier comprisesgreater than two types of immunostimulatory agents, wherein one or moretypes of immunostimulatory agents stimulate B cells, and one or moretypes of immunostimulatory agents stimulate T cells.

In some embodiments, various assays can be utilized in order todetermine whether an immune response has been modulated in a B cell orgroup of B cells or in a T cell or group of T cells. In someembodiments, the assay assesses whether or not the cell or group ofcells has/have become “activated”.

In some embodiments, various assays can be utilized in order todetermine whether an immune response has been stimulated in a T cell orgroup of T cells. In some embodiments, stimulation of an immune responsein T cells can be determined by measuring antigen-induced production ofcytokines by T cells. In some embodiments, stimulation of an immuneresponse in T cells can be determined by measuring antigen-inducedproliferation of T cells. In some embodiments, an immune response in Tcells is determined to be stimulated if cellular markers of T cellactivation are expressed at different levels (e.g. higher or lowerlevels) relative to unstimulated cells.

In some embodiments, various assays can be utilized in order todetermine whether an immune response has been stimulated in a B cell orgroup of B cells. In some embodiments, stimulation of an immune responsein B cells can be determined by measuring antibody titers, antibodyaffinities, antibody performance in neutralization assays, class-switchrecombination, affinity maturation of antigen-specific antibodies,development of memory B cells, development of long-lived plasma cellsthat can produce large amounts of high-affinity antibodies for extendedperiods of time, germinal center reactions, and/or antibody performancein neutralization assays.

A vaccine nanocarrier is an entity that comprises an immunofeaturesurface. The vaccine nanocarrier may also comprise at least oneimmunomodulatory agent which is capable of stimulating an immuneresponse in B cells and/or T cells. Any vaccine nanocarrier can be usedin accordance with the present invention.

In some embodiments, a nanocarrier has a greatest dimension (e.g.,diameter) of less than 100 microns (μm). In some embodiments, inventivenanocarriers have a greatest dimension (e.g., diameter) of 300 nm orless. In some embodiments, inventive nanocarriers have a greatestdimension (e.g., diameter) of 250 nm or less. In some embodiments,inventive nanocarriers have a greatest dimension (e.g., diameter) of 200nm or less. In some embodiments, inventive nanocarriers have a greatestdimension (e.g., diameter) of 150 nm or less. In some embodiments,inventive nanocarriers have a greatest dimension (e.g., diameter) of 100nm or less. In some embodiments, inventive nanocarriers have a greatestdimension ranging between 25 nm and 200 nm. In some embodiments,inventive nanocarriers have a greatest dimension ranging between 20 nmand 100 nm.

A variety of different nanocarriers can be used in accordance with thepresent invention. In some embodiments, nanocarriers are spheres orspheroids. In some embodiments, nanocarriers are flat or plate-shaped.In some embodiments, nanocarriers are cubes or cuboids. In someembodiments, nanocarriers are ovals or ellipses. In some embodiments,nanocarriers are cylinders, cones, or pyramids. Nanocarriers compriseone or more surfaces, and at least one of the one or more surfacescomprises an immunofeature surface. Nanocarriers may be solid or hollowand may comprise one or more layers. In some embodiments, each layer hasa unique composition and unique properties relative to the otherlayer(s). To give but one example, nanocarriers may have a core/shellstructure, wherein the core is one layer (e.g. a polymeric core) and theshell is a second layer (e.g. a lipid bilayer or monolayer).Nanocarriers may comprise a plurality of different layers. In someembodiments, one layer may be substantially cross-linked, a second layeris not substantially cross-linked, and so forth. In some embodiments,one, a few, or all of the different layers may comprise one or moreimmunomodulatory agents, targeting moieties, immunostimulatory agents,and/or combinations thereof. In some embodiments, one layer comprises animmunomodulatory agent, targeting moiety, and/or immunostimulatoryagent, a second layer does not comprise an immunomodulatory agent,targeting moiety, and/or immunostimulatory agent, and so forth. In someembodiments, each individual layer comprises a differentimmunomodulatory agent, targeting moiety, immunostimulatory agent,and/or combination thereof.

In some embodiments, nanocarriers may optionally comprise one or morelipids. In some embodiments, a nanocarrier is a liposome. In someembodiments, a nanocarrier comprises a lipid bilayer, and/or multiplelipid bilayers. For example, a lipid bilayer may form the exteriorsurface of a nanocarrier, in which case the nanocarrier comprising alipid bilayer shell may be referred to as a liposome. Liposomenanocarriers typically have relatively moldable surfaces, and thenanocarriers may take on a variety of shapes (e.g., spherical, oblong,cylindrical, etc.) depending on environmental factors. It will beappreciated, therefore, that the maximum diameter of such nanocarriersmay change in different environments. Typically, liposome nanocarrierscomprise phospholipids. In some embodiments, a nanocarrier comprises alipid monolayer. In some embodiments, a nanocarrier is a micelle. Insome embodiments, a nanocarrier comprises a core of a polymeric matrixsurrounded by a lipid layer (e.g. lipid bilayer, lipid monolayer, etc.).In some embodiments, a nanocarrier comprises a non-polymeric core (e.g.metal particle, quantum dot, ceramic particle, bone particle, viralparticle, etc.) surrounded by a lipid layer (e.g. lipid bilayer, lipidmonolayer, etc.).

In some embodiments, a nanocarrier comprises one or more polymers. Insome embodiments, a polymeric matrix can be surrounded by a coatinglayer (e.g. liposome, lipid monolayer, micelle, etc.). In someembodiments, an immunomodulatory agent, targeting moiety, and/orimmunostimulatory agent can be associated with the polymeric matrix. Insuch embodiments, the immunomodulatory agent, targeting moiety, and/orimmunostimulatory agent is effectively encapsulated within thenanocarrier. It will be appreciated, however, that the plurality ofmoieties on the immunofeature surface (i.e., that provide targeting toAPCs) are on a surface of the nanocarriers, the surface being anexterior surface and exposed to the environment surrounding thenanocarriers.

In some embodiments, an immunomodulatory agent, targeting moiety, and/orimmunostimulatory agent can be covalently associated with a nanocarrier.The targeting moieties mentioned here (and as described in more detailherein) are, for example, B-cell targeting moieties or T-cell targetingmoieties. It will be appreciated that such moieties are in addition tothe plurality of moieties that are present on the immunofeature surfaceand that provide targeting of the nanocarriers to APCs. In someembodiments, covalent association is mediated by a linker. In someembodiments, an immunomodulatory agent, targeting moiety, and/orimmunostimulatory agent is non-covalently associated with a nanocarrier.For example, in some embodiments, an immunomodulatory agent, targetingmoiety, and/or immunostimulatory agent is encapsulated within,surrounded by, and/or dispersed throughout a polymeric matrix, a lipidmembrane, etc. Alternatively or additionally, an immunomodulatory agent,targeting moiety, and/or immunostimulatory agent may be associated witha polymeric matrix, a lipid membrane, etc. by hydrophobic interactions,charge interactions, van der Waals forces, etc.

A wide variety of polymers and methods for forming polymeric matricestherefrom are known in the art of drug delivery. In general, a polymericmatrix comprises one or more polymers. Any polymer may be used inaccordance with the present invention. Polymers may be natural orunnatural (synthetic) polymers. Polymers may be homopolymers orcopolymers comprising two or more monomers. In terms of sequence,copolymers may be random, block, or comprise a combination of random andblock sequences. Polymers in accordance with the present invention maybe organic polymers. In some embodiments, the polymers are dendriticpolymers or blends of polymers.

Examples of polymers include polyethylenes, polycarbonates (e.g.poly(1,3-dioxan-2one)), polyanhydrides (e.g. poly(sebacic anhydride)),polyhydroxyacids (e.g. poly(β-hydroxyalkanoate)), polypropylfumerates,polycaprolactones, polyamides (e.g. polycaprolactam), polyacetals,polyethers, polyesters (e.g. polylactide, polyglycolide),poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polyureas, polystyrenes, and polyamines.

In some embodiments, nanocarriers comprise immunomodulatory agentsembedded within reverse micelles. To give but one example, a liposomenanocarrier may comprise hydrophobic immunomodulatory agents embeddedwithin the liposome membrane, and hydrophilic immunomodulatory agentsembedded with reverse micelles found in the interior of the liposomalnanocarrier.

In some embodiments, a nanocarrier does not include a polymericcomponent. In some embodiments, nanocarriers comprise metal particles,quantum dots, ceramic particles, bone particles, viral particles, etc.In some embodiments, an immunomodulatory agent, targeting moiety, and/orimmunostimulatory agent is associated with the surface of such anon-polymeric nanocarrier. In some embodiments, a non-polymericnanocarrier is an aggregate of non-polymeric components, such as anaggregate of metal atoms (e.g. gold atoms). In some embodiments, animmunomodulatory agent, targeting moiety, and/or immunostimulatory agentis associated with the surface of, encapsulated within, surrounded by,and/or dispersed throughout an aggregate of non-polymeric components.

In some embodiments, nanocarriers may optionally comprise one or moreamphiphilic entities (i.e., entities that possess both hydrophilic andhydrophobic properties). In some embodiments, an amphiphilic entity canpromote the production of nanocarriers with increased stability,improved uniformity, or increased viscosity.

In some embodiments, a nanocarrier comprises one or more nanoparticlesassociated with the exterior surface of and/or encapsulated within thenanocarrier.

Nanocarriers may be prepared using any method known in the art. Forexample, particulate nanocarrier formulations can be formed by methodssuch as nanoprecipitation, flow focusing fluidic channels, spray drying,single and double emulsion solvent evaporation, solvent extraction,phase separation, milling, microemulsion procedures, nanoprinting,microfabrication, nanofabrication, sacrificial layers, simple andcomplex coacervation, as well as other methods well known to those ofordinary skill in the art. Alternatively or additionally, aqueous andorganic solvent syntheses for monodisperse semiconductor, conductive,magnetic, organic, and other nanoparticles may be utilized.

In some embodiments, immunofeature moieties, immunomodulatory agents,targeting moieties, and/or immunostimulatory agents, are not covalentlyassociated with a nanocarrier. For example, nanocarriers may comprise apolymeric matrix, and immunomodulatory agents, targeting moieties,and/or immunostimulatory agents, etc. are associated with the surfaceof, encapsulated within, and/or distributed throughout the polymericmatrix of an inventive nanocarrier. Immunomodulatory agents may bereleased by diffusion, degradation of the nanocarrier, and/or acombination thereof. In some embodiments, polymer(s) of the nanocarrierdegrade by bulk erosion. In some embodiments, polymer(s) of thenanocarrier degrade by surface erosion.

In some embodiments, immunomodulatory agents, targeting moieties, and/orimmunostimulatory agents are covalently associated with a particle. Insome embodiments, covalent association is mediated by one or morelinkers. Any suitable linker can be used in accordance with the presentinvention. In some embodiments, the linker is a cleavable linker (e.g.,an ester linkage, an amide linkage, a disulfide linkage, etc.).

In some embodiments, nanocarriers are made by self-assembly. As anexample, lipids are mixed with a lipophilic immunomodulatory agent, andthen formed into thin films on a solid surface. A hydrophilicimmunomodulatory agent is dissolved in an aqueous solution, which isadded to the lipid films to hydrolyze lipids under vortex. Liposomeswith lipophilic immunomodulatory agents incorporated into the bilayerwall and hydrophilic immunomodulatory agents inside the liposome lumenare spontaneously assembled. In certain embodiments, pre-formulatedpolymeric nanoparticles are mixed with small liposomes under gentlevortex to induce liposome fusion onto polymeric nanoparticle surface.

As another example, a hydrophilic immunomodulatory agent to beencapsulated is first incorporated into reverse micelles by mixing withnaturally derived and non-toxic amphiphilic entities in a volatile,water-miscible organic solvent. In some embodiments, a biodegradablepolymer is added after reverse micelle formation is complete. Theresulting biodegradable polymer-reverse micelle mixture is combined witha polymer-insoluble hydrophilic non-solvent to form nanoparticles by therapid diffusion of the solvent into the non-solvent and evaporation ofthe organic solvent.

In some embodiments, lipid monolayer stabilized polymeric nanocarriersare used to deliver one or a plurality of immunomodulatory agents. Incertain embodiments, a hydrophilic immunomodulatory molecule is firstchemically conjugated to the polar headgroup of a lipid. The conjugateis mixed with a certain ratio of unconjugated lipid molecules in anaqueous solution containing one or more water-miscible solvents. Abiodegradable polymeric material is mixed with the hydrophobicimmunomodulatory agents to be encapsulated in a water miscible orpartially water miscible organic solvent. The resulting polymer solutionis added to the aqueous solution of conjugated and unconjugated lipid toyield nanoparticles by the rapid diffusion of the organic solvent intothe water and evaporation of the organic solvent.

The compositions and methods described herein can be used for theprophylaxis and/or treatment of a variety of infectious diseases,disorders, and/or conditions. Examples of other diseases, disorders,and/or conditions are provided elsewhere herein. In some embodiments,vaccine nanocarriers in accordance with the present invention may beused to treat, alleviate, ameliorate, relieve, delay onset of, inhibitprogression of, reduce severity of, and/or reduce incidence of one ormore symptoms or features of a disease, disorder, and/or condition. Insome embodiments, inventive vaccine nanocarriers may be used to treat,alleviate, ameliorate, relieve, delay onset of, inhibit progression of,reduce severity of, and/or reduce incidence of one or more symptoms orfeatures of microbial infection (e.g. bacterial infection, fungalinfection, viral infection, parasitic infection, etc.). In someembodiments, the prophylaxis and/or treatment of microbial infectioncomprises administering a therapeutically effective amount of inventivevaccine nanocarriers to a subject in need thereof, in such amounts andfor such time as is necessary to achieve the desired result. In certainembodiments of the present invention, a “therapeutically effectiveamount” of an inventive vaccine nanocarrier is that amount effective fortreating, alleviating, ameliorating, relieving, delaying onset of,inhibiting progression of, reducing severity of, and/or reducingincidence of one or more symptoms or features of disease, disorder,and/or condition provided herein.

In some embodiments, inventive prophylactic and/or therapeutic protocolsinvolve administering a therapeutically effective amount of one or moreinventive vaccine nanocarriers to a subject such that an immune responseis modulated (e.g., stimulated in both T cells and/or B cells).

The present invention provides novel compositions comprising atherapeutically effective amount of one or more vaccine nanocarriers andone or more pharmaceutically acceptable excipients. In some embodiments,the present invention provides for pharmaceutical compositionscomprising inventive vaccine nanocarriers as described herein. Thecomposition may include more than one type of nanocarrier, each typehaving different constituents (e.g., immunomodulatory agents, targetingagents, immunostimulatory agents, excipients, etc.). In accordance withsome embodiments, a method of administering a pharmaceutical compositioncomprising inventive compositions to a subject (e.g. human) in needthereof is provided.

In some embodiments, a therapeutically effective amount of an inventivevaccine nanocarrier composition is delivered to a patient and/or animalprior to, simultaneously with, and/or after diagnosis with a disease,disorder, and/or condition. In some embodiments, a therapeutic amount ofan inventive vaccine nanocarrier composition is delivered to a patientand/or animal prior to, simultaneously with, and/or after onset ofsymptoms of a disease, disorder, and/or condition. In certainembodiments, a therapeutic amount of an inventive vaccine nanocarriercomposition is administered to a patient and/or animal prior to exposureto an infectious agent. In certain embodiments, a therapeutic amount ofan inventive vaccine nanocarrier composition is administered to apatient and/or animal after exposure to an infectious agent. In certainembodiments, a therapeutic amount of an inventive vaccine nanocarriercomposition is administered to a patient and/or animal prior to exposureto an addictive substance or a toxin. In certain embodiments, atherapeutic amount of an inventive vaccine nanocarrier composition isadministered to a patient and/or animal after exposure to an addictivesubstance or a toxin.

In some embodiments, the pharmaceutical compositions of the presentinvention are administered by a variety of routes, including oral,intravenous, intramuscular, intra-arterial, intramedullary, intrathecal,subcutaneous, intraventricular, transdermal, interdermal, rectal,intravaginal, intraperitoneal, topical (as by powders, ointments,creams, and/or drops), transdermal, mucosal, nasal, buccal, enteral,sublingual; by intratracheal instillation, bronchial instillation,and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol.In certain embodiments, the composition is administered orally. Incertain embodiments, the composition is administered parenterally. Incertain embodiments, the composition is administered via intramuscularinjection.

In certain embodiments, vaccine nanocarriers which delay the onsetand/or progression of a disease, disorder, and/or condition (e.g., aparticular microbial infection) may be administered in combination withone or more additional therapeutic agents which treat the symptoms ofthe disease, disorder, and/or condition. For example, the vaccinenanocarriers may be combined with the use of an anti-cancer agent,anti-inflammatory agent, antibiotic, or anti-viral agent.

The invention provides a variety of kits comprising one or more of thenanocarriers of the invention. For example, the invention provides a kitcomprising an inventive nanocarrier and instructions for use. A kit maycomprise multiple different nanocarriers. A kit may comprise any of anumber of additional components or reagents in any combination.According to certain embodiments of the invention, a kit may include,for example, (i) a nanocarrier comprising at least one immunomodulatoryagent, wherein the at least one immunomodulatory agent is capable ofstimulating both a T cell and/or B cell response, at least one targetingmoiety, and/or at least one immunostimulatory agent; (ii) instructionsfor administering the nanocarrier to a subject in need thereof. Incertain embodiments, a kit may include, for example, (i) at least oneimmunomodulatory agent, wherein the at least one immunomodulatory agentis capable of stimulating both a T cell and B cell response; (ii) atleast one targeting moiety; (iii) at least one immunostimulatory agent;(iv) a polymeric matrix precursor; (v) lipids and amphiphilic entities;(vi) instructions for preparing inventive vaccine nanocarriers fromindividual components (i)-(v).

In some embodiments, the kit comprises an inventive nanocarrier andinstructions for mixing. Such kits, in some embodiments, also include animmunostimulatory agent and/or an antigen. The nanocarrier of such kitsmay comprise an immunomodulatory agent (e.g., a T cell antigen, such asa universal T cell antigen) and/or a targeting moiety. The T cellantigen and/or the targeting moiety may be on the surface of thenanocarrier. In some embodiments, the immunomodulatory agent and theantigen are the same. In some embodiments, they are different.

In some embodiments, the invention provides a composition comprising:(1) synthetic nanocarriers having at least one surface, wherein a firstsurface of the synthetic nanocarriers comprises an immunofeature surfacecomprising a plurality of moieties selected from nicotine or aderivative thereof; (2) an immunostimulatory agent, wherein theimmunostimulatory agent: (i) is associated with the immunofeaturesurface: (ii) is associated with a second surface of the nanocarrier; or(iii) is encapsulated within the core region of the nanocarrier, and (3)a pharmaceutically acceptable excipient. In some embodiments, theinvention provides a method comprising administering at least one doseof such a composition to a subject.

In some embodiments of the foregoing, an immunofeature surface comprisesa plurality of moieties present in an amount effective to provide lowaffinity and high avidity binding of the immunofeature surface to anantigen presenting cell.

In any of the foregoing embodiments described above, the word conjugatedmeans covalently or noncovalently conjugated, unless the context clearlyindicates otherwise. In any of the foregoing embodiments describedabove, the word encapsulated means physically trapped within, whether byadmixture, by a shell surrounding a core, by covalent bonding internalof the surface of the nanocarrier, and the like.

This application refers to various issued patents, published patentapplications, journal articles, and other publications, all of which areincorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1: Combined vaccine targeting strategy for optimal humoral andcellular immune response. The composite vaccine carries internal T cellantigens, adjuvants (not shown) and targeting moieties for DCs, FDC andSCS-Mph together with surface antigen for B cell recognition. Upon s.c.or i.m. injection, the material reaches lymph nodes via draining lymphvessels and accumulates on each APC (for clarity, only APC-specifictargeting moieties are shown, but each APC acquires the entire complex).DCs internalize and digest the complex and present antigenic peptides inMHC class I and class II to CD8 and CD4 T cells, respectively. Theactivated T cells differentiate into effector/memory (T_(Eff/Mem)) cellsthat mediate cellular immune responses. T_(FH) cells provide help to Bcells that were initially stimulated by antigen on SCS-Mph and in theprocess have acquired and processed T cell antigens for restimulation ofT_(FH). The help provided by T_(FH) cells allows the development of a GCreaction during which B cells proliferate and generate high-affinityantibodies.

FIG. 2: SCS-Mph bind lymph-borne viral particles and present them tofollicular B cells. (A) Immunohistochemical staining of the cortex of amouse popliteal lymph node stained with anti-CD169 and counter-stainedwith wheat germ agglutinin. The lymph node was harvested 30 minutesafter footpad injection of red fluorescent vesicular stomatitis virus(VSV). In the subcapsular sinus of the draining lymph node, the redvirus colocalized exclusively with CD169⁺ macrophages. (B) Electronmicrograph of a lymph node macrophage (Mph) and a follicular B cell (B1)below the floor of the subcapsular sinus (SCSf) 30 minutes after VSVinjection shows VSV at the surface and within a phagolysosome of the Mphand at the interface between Mph and B cells (arrowheads). (C) Injectionof VSV into the footpad of untreated mice (B6) results in rapiddownregulation of surface-expressed IgM on virus-specific B cells, asign of B cell activation. Depletion of SCS-Mph after footpad injectionof clodronate liposomes (CLL) abolished B cell activation, indicatingthat SCS-Mph are essential to present particulate antigen to B cells.

FIG. 3: An exemplary liposome nanocarrier with a lipophilicimmunomodulatory agent incorporated in the membrane, and a hydrophilicimmunomodulatory agent encapsulated within the liposome.

FIG. 4: An exemplary nanoparticle-stabilized liposome nanocarrier with alipophilic immunomodulatory agent incorporated into the membrane, and ahydrophilic immunomodulating agent encapsulated within the liposome.

FIG. 5: An exemplary liposome-polymer nanocarrier with a lipophilicimmunomodulatory agent incorporated into the membrane, and a hydrophobicimmunomodulating agent encapsulated within the polymeric nanoparticle.

FIG. 6: An exemplary nanoparticle-stabilized liposome-polymernanocarrier with a lipophilic immunomodulatory agent incorporated intothe membrane, and a hydrophobic immunomodulating agent encapsulatedwithin the polymeric nanoparticle.

FIG. 7: An exemplary liposome-polymer nanocarrier containing reversemicelles with a lipophilic immunomodulatory agent incorporated into themembrane, and a hydrophilic immunomodulatory agent encapsulated withinthe reverse micelles.

FIG. 8: An exemplary nanoparticle-stabilized liposome-polymernanocarrier containing reverse micelles with a lipophilicimmunomodulatory agent incorporated into the membrane, and a hydrophilicimmunomodulatory agent encapsulated inside the liposome.

FIG. 9: An exemplary lipid-stabilized polymeric nanocarrier with ahydrophilic immunomodulatory agent conjugated to the lipid monolayer,and a hydrophobic immunomodulatory agent encapsulated inside the polymercore.

FIG. 10: An exemplary lipid-stabilized polymeric nanocarrier containingreverse micelles with a hydrophilic immunomodulatory agent conjugated tothe lipid monolayer, and a hydrophilic immunomodulatory agentencapsulated inside the polymer core.

FIG. 11: Capture of lymph-borne VSV by SCS macrophages. (A) MP-IVMmicrographs of VSV in a popliteal LN (numbers: minutes after footpadinjection; scale bar: 100 μm). (B) VSV accumulation in aC57BL/6→Act(EGFP) recipient 3 hours after injection (scale bar: 50 μm).(C) Electron micrographs of VSV in LN 5 minutes after injection. Centermicrograph is shown schematically (left) and at higher magnification(right). Arrowheads identify VSV particles (scale bars: 2 μm). (D)Confocal micrographs of VSV-draining LN (30 minutes). Scale bars: 100 μm(left), 15 μm (right). (E) VSV titers in popliteal LNs 2 hours afterinjection into wildtype, C3-deficient or CLL-depleted mice. ***: p<0.001(two-way ANOVA, Bonferroni's post-test). (F) VSV capture in DH-LMP2amice. *: p<0.05 (unpaired t-test). (G) VSV titers after footpadinjection in untreated and CLL-treated mice (one of two similarexperiments; n=3). ProxLN: inguinal, paraaortic LNs; BrachLN: brachialLN. (H) Viral titers in lymph, spleen and blood after TD cannulation; *:p<0.05 (unpaired t-test). Horizontal bars in (E-H) indicate means.

FIG. 12: Characterization of CD169⁺ macrophages in peripheral LNs. (A-C)Lineage marker expression analysis of pooled mononuclear cells from LNsof naïve C57BL/6 mice. (A) After gating on the CD169⁺ population (middlepanel), cells were analyzed for expression of the twomacrophage-associated surface markers, I-Ab (MHC class II) and CD11b(bottom panel). Staining with an isotype control for anti-CD169 is shownin the top panel. (B) CD169⁺I-Ab⁺CD11b⁺ cells were further analyzed forexpression of CD68, F4/80, CD11c, and Gr-1. Gates were drawn to identifymarker+ cells, except for CD11c staining where the marker was positionedto identify conventional CD11c^(high) dendritic cells (overlay). Numbersindicate percentage of CD169⁺I-Ab⁺CD11b⁺ cells under the histogram gate.Data are representative of 3-5 experiments with similar results. (C)Quantitative analyses of data in panel (B), error bars represent SEM.(D-G) Confocal micrographs of popliteal LNs from naïve C57BL/6 miceshowing co-expression of selected markers on CD169⁺ cells (arrowheads).Scale bars: 125 μm in the left column and 20 μm in all other columns.

FIG. 13: Morphological changes in popliteal LNs following CLL treatment.(A) Confocal micrographs of popliteal LNs (top three rows) and spleens(bottom row) of untreated control mice (−CLL, left column) and animalsthat had received CLL footpad injections 6-10 days earlier. CLLtreatment depleted CD169⁺ macrophages in the LN (top row), but not inspleens; Lyve-1⁺ medullary lymphatic endothelial cells (second row) andcortical CD11c^(high) dendritic cells (third row) were not affected. (B)Cellular subset frequency in popliteal LNs with and without CLLtreatment, data are from n=3 mice and shown as mean±SEM; *: p<0.05, **:p<0.01; unpaired student's t-test. (C) Frequency of differentI-Ab⁺CD11b⁺ leukocyte subsets in popliteal LNs at 6-10 days afterfootpad injection of 50 μl CLL. Each symbol represents pooled poplitealLNs from one mouse. Subset frequencies among total mononuclear cells inpopliteal LNs were assessed by flow cytometry after gating onI-Ab+CD11b+ cells as shown in FIG. 12A. (D) Immunohistochemical analysisof popliteal LNs without treatment (−CLL) or 7 days after footpadinjection of CLL (+CLL). Scale bars: 300 mm. (E) Ultrastructure of theSCS in a representative popliteal LN 7 days after CLL treatment and 5minutes after footpad injection of 20 μg VSV-IND. Note the completeabsence of SCS macrophages and viral particles. Scale bar: 2 μm.

FIG. 14: Retention of fluorescent viruses and latex nanoparticles inpopliteal LNs. (A) Confocal micrographs of popliteal LNs 30 minutesafter footpad injection of Alexa-568-labeled adenovirus (AdV). Frozensections were stained with FITC-α-CD169 and Alexa-647-α-B220 to identifyB cells. Scale bars: 100 μm (left panel) and 15 μm (right panel). (B)Transmission electron micrographs of AdV particles captured by a SCSmacrophage. The top panel shows an annotated schematic drawing of thelow magnification overview (middle panel). The boxed area in the middlepanel is enlarged in the lower panel, arrowheads denote electron-dense,spherical AdV particles. Scale bars: 2 μm (top and middle panel) and 1μm (lower panel). (C-D) Confocal micrographs of popliteal LNs fromC57BL/6 mice 30 minutes after footpad injection of 20 μg Alexa-568labeled UV-inactivated AdV (C) or VV (D). Fluorescent virusesaccumulated in the cortical SCS above B follicles identified byFITC-α-B220 staining and also in the medulla where viruses were not onlybound by CD169⁺ macrophages, but also by LYVE-1⁺ lymphatic endothelialcells. Scale bars indicate 125 μm (left panel) and 25 μm (right panel).(E) Confocal micrograph of a popliteal LN 30 minutes after hind footpadinjection of Alexa-568 labeled VSV and approximately 10¹¹ CrimsonFluospheres (200 nm diameter). Frozen LN sections were counter-stainedwith FITC-α-CD169. Note that the Latex beads, unlike VSV, were poorlyretained in draining LNs. Scale bar: 125 μm.

FIG. 15: Effect of CLL footpad injection on VSV distribution in drainingLNs. Confocal micrographs show the localization of fluorescent VSVparticles in popliteal LNs without (A) or 7 days after (B) CLLtreatment. B follicles were identified by FITC-α-B220 staining. In themedulla (boxed area), VSV was bound by LYVE-1⁺ cells that were notaffected by CLL treatment. Scale bars: 125 μm (left column) and 25 μm(right column).

FIG. 16: SCS macrophages present lymph-derived AdV to follicular Blymphocytes. (A) Confocal micrograph of CD169⁺ macrophages in the SCSabove a B follicle in a popliteal LN. Frozen sections werecounterstained with wheatgerm agglutinin (WGA) to identify extracellularmatrix and with α-B220 to detect B cells. Note that some B cells residein the SCS, and one B cell appears to migrate between the follicle andthe SCS (arrowhead). Scale bar: 25 μm. (B) Electron micrograph and (C)schematic drawing of a SCS macrophage and surrounding cells in apopliteal LN 30 minutes after footpad injection of AdV. Scale bar: 2 μm.The boxes drawn in (C) indicate areas of higher magnification shown inpanels (D) and (E). These panels show two examples of AdV particles atthe interface between the SCS macrophage and B cells (arrowheads).Asterisks denote other macrophage-associated AdV particles. Scale bars:500 nm.

FIG. 17: Macrophage-mediated transfer of lymph-borne VSV across the SCSfloor alters virus-specific B cell behavior. (A) Electron micrographsand schematic drawing (middle) showing a macrophage penetrating the SCSfloor of a popliteal LN 30 minutes after VSV injection. Scale bars: 10μm (left) and 2 μm (right). Arrow: vacuole with digested VSV.Arrowheads: virions in contact zone between macrophage and B cells. (B)MP-IVM of polyclonal and VI10YEN B cells in popliteal LNs. Scale bars:50 μm. (C) Regional ratios of VI10YEN B cells/control B cells followingVSV injection. Results are from 3 movies/group. (D,E) Localization ofVI10YEN B cells in popliteal LNs relative to the SCS. **: p<0.01(one-way ANOVA with Bonferroni's post-test).

FIG. 18: Characteristics of VSV serotypes and VSV-IND-specific VI10YEN Bcells. (A) SDS-PAGE gels (12%) of purified VSV lysates. Top: VSV-IND andVSV-NJ. The N and P proteins co-migrate in VSV-NJ, approximate molecularweights are shown in parentheses. (B) Binding of Alexa-488 labeledVSV-IND (middle row) or VSV-NJ (bottom row) to B cells from C57BL/6 mice(left column) or VI10YEN mice (right column). The upper row showscontrol staining with the anti-idiotypic antibody 35.61 to the VI10YENBCR (Dang and Rock, 1991, J. Immunol., 146:3273). (C) Intracellularcalcium flux in CD43^(neg) purified, Fluo-LOJO loaded B cells fromVI10YEN mice (upper row) or C57BL/6 mice (lower row). Events werecollected continuously over time, asterisks indicate the timepoint whenantibodies or virus were added. Virus particles were used at 1000/Bcell, anti-IgM-(Fab)₂ at 10 ng/10⁶ B cells. (D) Neutralization assay fortotal Ig and IgG in serum of C57BL/6 mice 4 and 10 days afterimmunization by footpad injection of 10 μg UV-VSV orUV-VSV-AlexaFluor-488-IND. (E) Calcium flux in VI10YEN B cells exposedto supernatant from VSV stocks. Supernatant was generated byultracentrifugation through a sucrose cushion resulting in approximately10,000-fold reduction in viral titers and was used on B cells eitherundiluted (top right) or at 1:100 dilution (bottom right). As a control,VSV stock solution was diluted to equivalent viral titers (MOI; leftpanels). The results demonstrate the presence of antigenic VSV-G that isnot associated with virus particles in our virus preparation.

FIG. 19: VSV-induced adhesion of VI10YEN B cells to ICAM-1 and VCAM-1.(A,B) Adhesion of purified naïve and VSV-IND activated (30 minuteexposure) VI10YEN B cells to plastic plates coated with the indicatedconcentrations of recombinant ICAM-1-Fc (A) or VCAM-1-Fc (B). Pooleddata of two triplicate experiments are shown. Horizontal bars representmeans. (C, D) Confocal micrographs of ICAM-1 and VCAM-1 expression inpopliteal LNs of C57BL/6 mice. Scale bars: 50 μm. (E) Adhesion ofpurified naïve wildtype and VI10YEN B cells to plastic dishes coatedwith the indicated pfu-equivalent concentrations of UV-inactivatedVSV-IND. Data represent means±SEM of triplicates.

FIG. 20: SCS macrophages are required for early activation ofVSV-specific B cells in LNs. (A) Confocal micrograph shows MHC-IIcolocalization with VSV-IND (30 minutes after injection) inVI10YENxMHCII(EGFP) B cells at the SCS (arrowhead), not the deepfollicle (asterisk). Scale bar: 25 μm. (B) Distance of VSV-associatedand VSV-free VI10YENxMHCII(EGFP) B cells to the SCS. Horizontal lines:medians. (C) BCR expression kinetics on VI10YEN and (D) polyclonal Bcells after VSV-IND footpad injection. (E) BCR expression on VI10YENcells in CLL-treated or untreated popliteal LNs after VSV-IND injection(20 μg). Mean fluorescence intensities were normalized to virus-freevalues (dashed line). Means±SEM (3-5 mice). (F) Confocal micrograph ofVI10YEN B cells in control and (G) CLL-treated popliteal LNs 6 hoursafter VSV-IND injection (0.4 μg). Scale bar: 125 μm. (H) VI10YEN B cellfrequency at T/B borders and in follicles 6 hours after VSV-INDinjection at indicated doses. Means±SEM; n=3-4 follicles/2 mice; *:p<0.05; **: p<0.01; ***: p<0.001 (t-test).

FIG. 21: VI10YEN B cell motility in draining LNs following virusinjection. Median 3D instantaneous velocities of wildtype (triangles)and VI10YEN B cells (circles) in deep follicles and the SCS/superficialfollicle about 5-35 min after VSV footpad injection. Horizontal barsrepresent means; *: p<0.05; **: p<0.01 (one-way ANOVA with Bonferroni'spost test). Note that specific B cells slow down throughout the entirefollicle, likely as a consequence of free VSV-G in our preparation (seeFIG. 18). Control experiments showed similar B cell motility parametersin CLL-treated and nontreated popLNs.

FIG. 22: Timecourse of activation marker induction on VI10YEN B cells invirus-draining and non-draining LNs following injection of VSV-IND.VI10YEN B cells were fluorescently tagged with CMTMR and transferred tonaïve mice that were injected 18 hours later with 20 μg UV-inactivatedVSV-IND (time 0 hours). The draining popliteal LN (popLN) and a distalbrachial LN (brachLN) were harvested after the indicated time intervalsto generate single-cell suspensions. CD69 and CD86 expression on B cellswas assessed by flow cytometry after gating on (A) B220⁺CMTMR⁺VI10YENcells or (B) B220⁺CMTMR⁻ endogenous control B cells.

FIG. 23: Confocal (left and middle columns) and MP-IVM micrographs(right column) of popliteal LNs of mice that had received adoptivetransfers of a mixture of CMTMR-labeled VI10YEN B cells and CMAC-labeledpolyclonal B cells (in the right column). On the following day, 20 μgUV-inactivated VSV-IND was injected in a footpad and the drainingpopliteal LNs were either surgically prepared for MP-IVM or harvestedfor confocal analysis of frozen sections at the indicated time points.MP-IVM images show that VSV-specific, but not polyclonal B cells madecontact with VSV in the SCS as early as 30 minutes after virusinjection. VI10YEN B cells relocated to the T/B border at 6 hoursfollowing injection. Scale bars: 150 μm in the left column and 25 μm inthe other columns.

FIG. 24: In vivo targeting of SCS-Mph using Fc fragments from human IgG.(A) The FACS histograms on the left document the binding of fluorescentPEG-PLGA nanoparticles (˜100 nm diameter) to lymph node macrophages. (B)Fc-nanoparticle (NP) targets SCS-Mph and follicular dendritic cells.

FIG. 25: Identification of the chemokine receptor CX3CR1 (fractalkinereceptor) on macrophages in lymph node subcapsular sinus (SCS), but notin macrophages in the medulla. The micrograph on the right is a 3Dprojection of a lymph node from a double-knockin mouse where greenfluorescent protein (GFP) is expressed in the CX3CR1 locus, while redfluorescent protein (RFP) reports the expression of another chemokinereceptor, CCR2. SCS-Mph are readily identifiable by their prominentgreen fluorescence, while medullary macrophages express primarily RFP.

FIG. 26: SCS-Mph express the chemokine receptor CX3CR1. The graph showsa FACS plot of a single cell suspension from a lymph node of a knockinmouse that was genetically engineered to express GFP from the CX3CR1locus. SCS-Mphs are identified by staining with a soluble receptor,CRFc, which binds macrophages in the SCS, but not the medulla. The CRFcnegative CX3CR1-expressing (i.e., GFP-high) cells are conventionaldendritic cells that express this chemokine receptor.

FIG. 27: Fluorescent micrographs of frozen sections from mouse popliteallymph nodes 24 h after footpad injection of 0.2 μm diameter Latex beadssurface modified with either amine (left and middle panel) or carboxymoieties (right panel). Both sets of beads were purchased fromInvitrogen (Cat. no. F8763 and F8805). Sections on left and right werecounterstained with anti-CD169. Images are oriented so that the medulla(weak, diffuse staining with anti-CD169) faces to the right and thesubcapsular sinus (SCS) region (bright anti-CD169) faces to the left.Note that the red amine modified particles prominently localize to theSCS, while blue carboxy modified beads are primarily retained in themedulla.

FIG. 28: (A) Antigen-bearing targeted nanoparticles are highlyimmunogenic and induce high antibody titers. (B) The induced immuneresponse elicited by nanoparticle vaccines confers potent protectionfrom a lethal dose of VSV.

FIG. 29: In vivo T cell activation by immunomodulatory nanoparticles.(A) Effect of NPs on CD4 T cell activation. (B) Effect of NPs on CD8 Tcell response mixed with CpG adjuvant (TLR9 agonist). (C) Effect ofco-encapsulated adjuvant on CD8 T cell activation.

FIG. 30: Shows an exemplary nicotine conjugation strategy.

FIG. 31: Shows an exemplary R848 conjugation strategy.

FIGS. 32 a and 32 b: Shows preparation of nanoparticles with conjugatednicotine using various compositions.

FIG. 33: Shows mouse anti-nicotine IgG titers and concentrations up to23 weeks after the mice have been administered various compositions.

FIG. 34: Shows mouse anti-nicotine IgG titers and concentrations 21 daysafter the mice have been administered various compositions.

DETAILED DESCRIPTION Definitions

Abused substance: As used herein, the term “abused substance” is anysubstance taken by a subject (e.g., a human) for purposes other thanthose for which it is indicated or in a manner or in quantities otherthan directed by a physician. In some embodiments, the abused substanceis a drug, such as an illegal drug. In certain embodiments, the abusedsubstance is an over-the-counter drug. In some embodiments, the abusedsubstance is a prescription drug. The abused substance, in someembodiments, is an addictive substance. In some embodiments, the abusedsubstance has mood-altering effects, and, therefore, includes inhalantsand solvents. In other embodiments, the abused substance is one that hasno mood-altering effects or intoxication properties, and, therefore,includes anabolic steroids. Abused substances include, but are notlimited to, cannabinoids (e.g., hashish, marijuana), depressants (e.g.,barbituates, benodiazepines, flunitrazepam (Rohypnol), GHB, methaqualone(quaaludes)), dissociative anesthetics (e.g., ketamine, PCP),hallucinogens (e.g, LSD, mescaline, psilocybin), opioids and morphinederivatives (e.g., codeine, fentanyl, heroin, morphine, opium),stimulants (amphetamine, cocaine, Ecstacy (MDMA), methamphetamine,methylphenidate (Ritalin), nicotine), anabolic steroids, and inhalants.In some embodiments, the abused substance for inclusion in a nanocarrieris the complete molecule or a portion thereof.

Addictive substance: As used herein, the term “addictive substance” is asubstance that causes obsession, compulsion, or physical dependence orpsychological dependence. In some embodiments, the addictive substanceis an illegal drug. In other embodiment, the addictive substance is anover-the-counter drug. In still other embodiments, the addictivesubstance is a prescription drug. Addictive substances include, but arenot limited to, cocaine, heroin, marijuana, methamphetamines, andnicotine. In some embodiments, the addictive substance for inclusion ina nanocarrier is the complete molecule or a portion thereof.

Administering or administration: (1) dosing a pharmacologically activematerial, such as an inventive composition, to a subject in a mannerthat is pharmacologically useful, (2) directing that such material bedosed to the subject in a pharmacologically useful manner, or (3)directing the subject to self-dose such material in a pharmacologicallyuseful manner.

Amino acid: As used herein, term “amino acid,” in its broadest sense,refers to any compound and/or substance that can be incorporated into apolypeptide chain. In some embodiments, an amino acid has the generalstructure H₂N—C(H)(R)—COOH. In some embodiments, an amino acid is anaturally-occurring amino acid. In some embodiments, an amino acid is asynthetic amino acid; in some embodiments, an amino acid is a D-aminoacid; in some embodiments, an amino acid is an L-amino acid. “Standardamino acid” or “natural amino acid” refers to any of the twenty standardL-amino acids commonly found in naturally occurring peptides.“Nonstandard amino acid” refers to any amino acid, other than thestandard amino acids, regardless of whether it is prepared syntheticallyor obtained from a natural source. As used herein, “non-natural aminoacid” encompasses chemically produced or modified amino acids, includingbut not limited to salts, amino acid derivatives (such as amides),and/or substitutions Amino acids, including carboxy- and/oramino-terminal amino acids in peptides, can be modified by methylation,amidation, acetylation, and/or substitution with other chemical groupsthat can change the peptide's circulating half-life without adverselyaffecting their activity. Amino acids may participate in a disulfidebond. The term “amino acid” is used interchangeably with “amino acidresidue,” and may refer to a free amino acid and/or to an amino acidresidue of a peptide. It will be apparent from the context in which theterm is used whether it refers to a free amino acid or a residue of apeptide.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans, at anystage of development. In some embodiments, “animal” refers to non-humananimals, at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). Insome embodiments, animals include, but are not limited to, mammals,birds, reptiles, amphibians, fish, and/or worms. In some embodiments, ananimal may be a transgenic animal, genetically-engineered animal, and/ora clone.

Antibody: As used herein, the term “antibody” refers to anyimmunoglobulin, whether natural or wholly or partially syntheticallyproduced. All derivatives thereof which maintain specific bindingability are also included in the term. The term also covers any proteinhaving a binding domain which is homologous or largely homologous to animmunoglobulin binding domain. Such proteins may be derived from naturalsources, or partly or wholly synthetically produced. An antibody may bemonoclonal or polyclonal. An antibody may be a member of anyimmunoglobulin class, including any of the human classes: IgG, IgM, IgA,IgD, and IgE. As used herein, the terms “antibody fragment” or“characteristic portion of an antibody” are used interchangeably andrefer to any derivative of an antibody which is less than full-length.An antibody fragment can retain at least a significant portion of thefull-length antibody's specific binding ability. Examples of suchantibody fragments include, but are not limited to, Fab, Fab′, F(ab′)2,scFv, Fv, dsFv diabody, and Fd fragments. Antibody fragment also includeFc fragments. An antibody fragment may be produced by any means. Forexample, an antibody fragment may be enzymatically or chemicallyproduced by fragmentation of an intact antibody and/or it may berecombinantly produced from a gene encoding the partial antibodysequence. Alternatively or additionally, an antibody fragment may bewholly or partially synthetically produced. An antibody fragment mayoptionally comprise a single chain antibody fragment. Alternatively oradditionally, an antibody fragment may comprise multiple chains whichare linked together, for example, by disulfide linkages. An antibodyfragment may optionally comprise a multimolecular complex. A functionalantibody fragment will typically comprise at least about 50 amino acidsand more typically will comprise at least about 200 amino acids.

Approximately: As used herein, the terms “approximately” or “about” inreference to a number are generally taken to include numbers that fallwithin a range of 5%, 10%, 15%, or 20% in either direction (greater thanor less than) of the number unless otherwise stated or otherwise evidentfrom the context (except where such number would be less than 0% orexceed 100% of a possible value).

Associated with: As used herein, the term “associated with” refers tothe state of two or more entities which are linked by a direct orindirect covalent or non-covalent interaction. In some embodiments, anassociation is covalent. In some embodiments, a covalent association ismediated by a linker moiety. In some embodiments, an association isnon-covalent (e.g., charge interactions, affinity interactions, metalcoordination, physical adsorption, host-guest interactions, hydrophobicinteractions, TT stacking interactions, hydrogen bonding interactions,van der Waals interactions, magnetic interactions, electrostaticinteractions, dipole-dipole interactions, etc.). For example, in someembodiments, an entity (e.g., immunomodulatory agent, targeting moiety,immunostimulatory agent, nanoparticle, etc.) may be covalentlyassociated with a vaccine nanocarrier. In some embodiments, an entity(e.g., immunomodulatory agent, targeting moiety, immunostimulatoryagent, nanoparticle, etc.) may be non-covalently associated with avaccine nanocarrier. For example, the entity may be associated with thesurface of, encapsulated within, surrounded by, and/or distributedthroughout a lipid bilayer, lipid monolayer, polymeric matrix, etc. ofan inventive vaccine nanocarrier. Where an entity may be referred to bya particular name in the free (i.e., non-conjugated) form, it will beappreciated that, unless specified otherwise, the entity may also bereferred to by the same name even when the entity is conjugated to asecond entity. For example, the name “(S)-(−)-nicotine” refers to thecompound 3-[(2S)-1-methylpyrrolidin-2-yl]pyridine. Similarly, the terms“(S)-(−)-nicotine,” “(S)-(−)-nicotine fragment,” “(S)-(−)-nicotinemoiety,” and “(S)-(−)-nicotine analog” also refer to the chemical moiety

wherein a bond to one atom (typically, although not necessarily, a bondto a H atom) is replaced with a bond to the second moiety.

Biocompatible: As used herein, the term “biocompatible” refers tosubstances that are not toxic to cells. In some embodiments, a substanceis considered to be “biocompatible” if its addition to cells in vivodoes not induce inflammation and/or other adverse effects in vivo. Insome embodiments, a substance is considered to be “biocompatible” if itsaddition to cells in vitro or in vivo results in less than or equal toabout 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about20%, about 15%, about 10%, about 5%, or less than about 5% cell death.

Biodegradable: As used herein, the term “biodegradable” refers tosubstances that are degraded under physiological conditions. In someembodiments, a biodegradable substance is a substance that is brokendown by cellular machinery. In some embodiments, a biodegradablesubstance is a substance that is broken down by chemical processes.

B cell antigen: As used herein, the term “B cell antigen” refers to anyantigen that is recognized by and triggers an immune response in a Bcell. In some embodiments, an antigen that is a B cell antigen is also aT cell antigen. In certain embodiments, the B cell antigen is not also aT cell antigen. In certain embodiments, when a nanocarrier, as providedherein, comprises both a B cell antigen and a T cell antigen, the B cellantigen and T cell antigen are not the same antigen, although each ofthe B cell and T cell antigens may be, in some embodiments, both a Bcell antigen and a T cell antigen. In other embodiments, the B cellantigen and T cell antigen of the nanocarrier are the same.

Cell type: As used herein, the term “cell type” refers to a form of cellhaving a distinct set of morphological, biochemical, and/or functionalcharacteristics that define the cell type. One of skill in the art willrecognize that a cell type can be defined with varying levels ofspecificity. For example, T cells and B cells are distinct cell types,which can be distinguished from one another but share certain featuresthat are characteristic of the broader “lymphocyte” cell type of whichboth are members. Typically, cells of different types may bedistinguished from one another based on their differential expression ofa variety of genes which are referred to in the art as “markers” of aparticular cell type or types (e.g., cell types of a particularlineage). In some embodiments, cells of different types may bedistinguished from one another based on their differential functions. A“cell type-specific marker” is a gene product or modified versionthereof that is expressed at a significantly greater level by one ormore cell types than by all or most other cell types and whoseexpression is characteristic of that cell type. Many cell type specificmarkers are recognized as such in the art.

Dosage form: a drug in a medium, carrier, vehicle, or device suitablefor administration to a subject. Examples of dosage forms are providedherein.

Hazardous environmental agent: As used herein, the term “hazardousenvironmental agent” refers to any hazardous substance found in theenvironment. Such substances are generally believed to pose a healthrisk. Hazardous environmental agents include substances that are thoughtto pose a health risk even though they may not actually pose a risk.Hazardous environmental agents include, but are not limited to, arsenic,lead, mercury, vinyl chloride, polychlorinated biphenyls, benzene,polycyclic aromatic hydrocarbons, cadmium, benzo(a)pyrene,benzo(b)fluoranthene, chloroform, DDT, P,P′-, aroclor 1254, aroclor1260, dibenzo(a,h)anthracene, trichloroethylene, dieldrin, chromiumhexavalent, and DDE, P,P′. In some embodiments, the hazardousenvironmental agent for inclusion in a nanocarrier is the completemolecule or a portion thereof.

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, etc., rather than within an organism (e.g.,animal, plant, and/or microbe).

In vivo: As used herein, the term “in vivo” refers to events that occurwithin an organism (e.g., animal, plant, and/or microbe).

Immunostimulatory agent: As used herein, the term “immunostimulatoryagent” refers to an agent that modulates an immune response to anantigen but is not the antigen or derived from the antigen. “Modulate”,as used herein, refers to inducing, enhancing, suppressing, directing,or redirecting an immune response. Such agents include immunostimulatoryagents that stimulate (or boost) an immune response to an antigen but,as defined above, is not the antigen or derived from the antigen.Immunostimulatory agents, therefore, include adjuvants. In someembodiments, the immunostimulatory agent is on the surface of thenanocarrier and/or is encapsulated within the nanocarrier. In someembodiments, the immunostimulatory agent on the surface of thenanocarrier is different from the immunostimulatory agent encapsulatedwithin the nanocarrier. In some embodiments, the nanocarrier comprisesmore than one type of immunostimulatory agent. In some embodiments, themore than one type of immunostimulatory agent act on different pathways.Examples of immunostimulatory agents include those provided elsewhereherein. In some embodiments, all of the immunostimulatory agents of asynthetic nanocarrier are identical to one another. In some embodiments,a synthetic nanocarrier comprises a number of different types ofimmunostimulatory agents. In some embodiments, a synthetic nanocarriercomprises multiple individual immunostimulatory agents, all of which areidentical to one another. In some embodiments, a synthetic nanocarriercomprises exactly one type of immunostimulatory agent. In someembodiments, a synthetic nanocarrier comprises exactly two distincttypes of immunostimulatory agents. In some embodiments, a syntheticnanocarrier comprises greater than two distinct types ofimmunostimulatory agents. In some embodiments, a synthetic nanocarriercomprises a lipid membrane (e.g., lipid bilayer, lipid monolayer, etc.),wherein at least one type of immunostimulatory agent is associated withthe lipid membrane. In some embodiments, at least one type ofimmunostimulatory agent is embedded within the lipid membrane. In someembodiments, at least one type of immunostimulatory agent is embeddedwithin the lumen of a lipid bilayer. In some embodiments, a syntheticnanocarrier comprises at least one type of immunostimulatory agent thatis associated with the interior surface of the lipid membrane. In someembodiments, at least one type of immunostimulatory agent isencapsulated within the lipid membrane of a synthetic nanocarrier. Insome embodiments, at least one type of immunostimulatory agent may belocated at multiple locations of a synthetic nanocarrier. One ofordinary skill in the art will recognize that the preceding examples areonly representative of the many different ways in which multipleimmunostimulatory agents may be associated with different locales ofsynthetic nanocarriers. Multiple immunostimulatory agents may be locatedat any combination of locales of synthetic nanocarriers.

Nicotine: Unless indicated otherwise, throughout this disclosure theterms “nicotine,” “nicotine moiety,” and “nicotine hapten” are usedinterchangeably and are intended to include nicotine per se (i.e.,(S)-(−)-, (R)-(−)-, or a combination thereof) as well as metabolites,derivatives, and analogues thereof. Metabolites of nicotine include anycompound that is the product of metabolic processing of nicotine, suchas cotinine, continine N′-oxide (CNO), 5′-hydroxycotinine (5HC),3′-hydroxycotinine (3HC), 5′-hydroxycotinine (5HC),5′-hydroxycotinine-N-oxide, 3′-hydroxycotinine glucuronide, norcotinine,nornicotine, nicotine-N′-oxide (NNO), (S)-nicotine-N—B-glucuronide(Nicotine-Gluc), and Cotinine-glucuronide (Cotinine-Gluc). Derivativesof nicotine include conjugates of nicotine covalently bonded to anotherspecies (such as a polymer, oligomer, or small molecule). Analoguesinclude, for example, nicotine wherein the N-methyl group has beenreplaced with a higher order alkyl group. Similarly, the term“anti-nicotine antibody” refers to an antibody typically created in abiological organism (such as an animal) that binds to nicotine and/ormetabolites, derivatives, or analogues thereof.

Nucleic acid: As used herein, the term “nucleic acid,” in its broadestsense, refers to any compound and/or substance that is or can beincorporated into an oligonucleotide chain. In some embodiments, anucleic acid is a compound and/or substance that is or can beincorporated into an oligonucleotide chain via a phosphodiester linkage.In some embodiments, “nucleic acid” refers to individual nucleic acidresidues (e.g., nucleotides and/or nucleosides). In some embodiments,“nucleic acid” refers to an oligonucleotide chain comprising individualnucleic acid residues. As used herein, the terms “oligonucleotide” and“polynucleotide” can be used interchangeably. In some embodiments,“nucleic acid” encompasses RNA as well as single and/or double-strandedDNA and/or cDNA. Furthermore, the terms “nucleic acid”, “DNA”, “RNA”,and/or similar terms include nucleic acid analogs, i.e., analogs havingother than a phosphodiester backbone. For example, the so-called“peptide nucleic acids,” which are known in the art and have peptidebonds instead of phosphodiester bonds in the backbone, are consideredwithin the scope of the present invention. The term “nucleotide sequenceencoding an amino acid sequence” includes all nucleotide sequences thatare degenerate versions of each other and/or encode the same amino acidsequence. Nucleotide sequences that encode proteins and/or RNA mayinclude introns. Nucleic acids can be purified from natural sources,produced using recombinant expression systems and optionally purified,chemically synthesized, etc. Where appropriate, e.g., in the case ofchemically synthesized molecules, nucleic acids can comprise nucleosideanalogs such as analogs having chemically modified bases or sugars,backbone modifications, etc. A nucleic acid sequence is presented in the5′ to 3′ direction unless otherwise indicated. The term “nucleic acidsegment” is used herein to refer to a nucleic acid sequence that is aportion of a longer nucleic acid sequence. In many embodiments, anucleic acid segment comprises at least 3, 4, 5, 6, 7, 8, 9, 10, or moreresidues. In some embodiments, a nucleic acid is or comprises naturalnucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine,deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine);nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine,pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine,C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine,C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine,7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,O(6)-methylguanine, and 2-thiocytidine); chemically modified bases;biologically modified bases (e.g., methylated bases); intercalatedbases; modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose,arabinose, and hexose); and/or modified phosphate groups (e.g.,phosphorothioates and 5′-N-phosphoramidite linkages).

Particle: As used herein, a “particle” refers to any entity having adiameter of less than 10 microns (μm). Typically, particles have alongest dimension (e.g., diameter) of 1000 nm or less. In someembodiments, particles have a diameter of 300 nm or less. Particlesinclude microparticles, nanoparticles, and picoparticles. In someembodiments, nanoparticles have a diameter of 200 nm or less. In someembodiments, nanoparticles have a diameter of 100 nm or less. In someembodiments, nanoparticles have a diameter of 50 nm or less. In someembodiments, nanoparticles have a diameter of 30 nm or less. In someembodiments, nanoparticles have a diameter of 20 nm or less. In someembodiments, nanoparticles have a diameter of 10 nm or less. In someembodiments, particles can be a matrix of polymers. In some embodiments,particles can be a non-polymeric particle (e.g., a metal particle,quantum dot, ceramic, inorganic material, bone, etc.). Particles mayalso be liposomes and/or micelles. As used herein, the term“nanoparticle” refers to any particle having a diameter of less than1000 nm.

Pharmaceutically acceptable excipient: a pharmacologically inactivesubstance added to an inventive composition to further facilitateadministration of the composition. Examples, without limitation, ofpharmaceutically acceptable excipients include calcium carbonate,calcium phosphate, various diluents, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Poorly immunogenic antigen: As used herein, the term “poorly immunogenicantigen” refers to an antigen that does not trigger any or a sufficientlevel of a desired immune response. “Sufficient”, as used herein, refersto the ability to elicit a detectable or protective immune response whenadministered in a composition that does not employ a nanocarrierdescribed herein, e.g., as free antigen mixed with adjuvant in theabsence of a nanocarrier. In some embodiments, the desired immuneresponse is to treat or prevent a disease or condition. In certainembodiments, the desired immune response is to alleviate one or moresymptoms of a disease or condition. Poorly immunogenic antigens include,but are not limited to, self antigens, small molecules, andcarbohydrates.

Self antigen: As used herein, the term “self antigen” refers to a normalsubstance in the body of an animal that when an immune response againstthe antigen within the animal is triggered, autoimmunity (e.g., anautoimmune disease) can result. A self antigen can be a protein orpeptide, lipoprotein, lipid, carbohydrate, or a nucleic acid. Thenucleic acid can be a DNA or RNA. Self antigens include, but are notlimited to enzymes, structural proteins, secreted proteins, cell surfacereceptors, and cytokines. In some embodiments, the self antigen is acytokine, and the cytokine is TNF, IL-1, or IL-6. In some embodiments,the self antigen is cholesteryl ester transfer protein (CETP), a serumprotein responsible for cholesterol transfer from high-densitylipoprotein (HDL) to low-density lipoprotein cholesterol (LDL), the Aβprotein associated with Alzheimer's, a proteolytic enzyme that processesthe pathological form of the Aβ protein, LDL associated withatherosclerosis, or a coreceptor for HIV-1. In some embodiments, theproteolytic enzyme that processes the pathological form of the Aβprotein is beta-secretase. In some embodiments, the LDL associated withatherosclerosis is oxidized or minimally modified. In some embodiments,the coreceptor for HIV-1 is CCR5.

Small molecule: In general, a “small molecule” is understood in the artto be an organic molecule that is less than about 2000 g/mol in size. Insome embodiments, the small molecule is less than about 1500 g/mol orless than about 1000 g/mol. In some embodiments, the small molecule isless than about 800 g/mol or less than about 500 g/mol. In someembodiments, small molecules are non-polymeric and/or non-oligomeric. Insome embodiments, small molecules are not proteins, peptides, or aminoacids. In some embodiments, small molecules are not nucleic acids ornucleotides. In some embodiments, small molecules are not saccharides orpolysaccharides.

Specific binding: As used herein, the term “specific binding” refers tonon-covalent physical association of a first and a second moiety whereinthe association between the first and second moieties is at least 2times as strong, at least 5 times as strong as, at least 10 times asstrong as, at least 50 times as strong as, at least 100 times as strongas, or stronger than the association of either moiety with most or allother moieties present in the environment in which binding occurs.Binding of two or more entities may be considered specific if theequilibrium dissociation constant, K_(d), is 10⁻³ M or less, 10⁻⁴M orless, 10⁻⁵M or less, 10⁻⁶M or less, 10⁻⁷M or less, 10⁻⁸ M or less, 10⁻⁹M or less, 10⁻¹⁰ M or less, 10⁻¹¹ M or less, or 10⁻¹² M or less underthe conditions employed, e.g., under physiological conditions such asthose inside a cell or consistent with cell survival. In someembodiments, specific binding can be accomplished by a plurality ofweaker interactions (e.g., a plurality of individual interactions,wherein each individual interaction is characterized by a K_(d) ofgreater than 10⁻³M). In some embodiments, specific binding, which can bereferred to as “molecular recognition,” is a saturable bindinginteraction between two entities that is dependent on complementaryorientation of functional groups on each entity. Examples of specificbinding interactions include aptamer-aptamer target interactions,antibody-antigen interactions, avidin-biotin interactions,ligand-receptor interactions, metal-chelate interactions, hybridizationbetween complementary nucleic acids, etc.

Subject: As used herein, the term “subject” or “patient” refers to anyorganism to which a composition of this invention may be administered,e.g., for experimental, diagnostic, and/or therapeutic purposes. Typicalsubjects include animals (e.g., mammals such as non-human primates, andhumans; avians; domestic household or farm animals such as cats, dogs,sheep, goats, cattle, horses and pigs; laboratory animals such as mice,rats and guinea pigs; rabbits; fish; reptiles; zoo and wild animals)and/or plants. Typically, “subjects” are animals, including mammals suchas humans and primates; and the like.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with, can be diagnosedwith, or displays one or more symptoms of the disease, disorder, and/orcondition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition has not been diagnosed with and/or may notexhibit symptoms of the disease, disorder, and/or condition. In someembodiments, a disease, disorder, and/or condition is associated with amicrobial infection (e.g., bacterial infection, viral infection, fungalinfection, parasitic infection, etc.). In some embodiments, anindividual who is susceptible to a microbial infection may be exposed toa microbe (e.g., by ingestion, inhalation, physical contact, etc.). Insome embodiments, an individual who is susceptible to a microbialinfection may be exposed to an individual who is infected with themicrobe. In some embodiments, an individual who is susceptible to amicrobial infection is one who is in a location where the microbe isprevalent (e.g., one who is traveling to a location where the microbe isprevalent). In some embodiments, an individual who is susceptible to adisease, disorder, and/or condition will develop the disease, disorder,and/or condition. In some embodiments, an individual who is susceptibleto a disease, disorder, and/or condition will not develop the disease,disorder, and/or condition. In some embodiments, the subject has or issusceptible to having cancer, an infectious disease, a non-autoimmunemetabolic or degenerative disease, or an addiction. In some embodiments,the subject has or is susceptible to having a bacterial, fungal,protozoan, parasitic, or viral infection. The cause of such infectioncan be any of the organisms as provided herein. In some embodiments, thesubject has or is susceptible to tuberculosis, malaria, leishmaniasis,H. pylori, a Staphylococcus infection, or a Salmonella infection. Insome embodiments, the subject has or is susceptible to having influenza.In some embodiments, the subject has or is susceptible to an autoimmunedisease.

Synthetic nanocarrier(s): “Synthetic nanocarrier(s)” means a discreteobject that is not found in nature, and that possesses at leastdimension that is less than or equal to 5 microns in size. Albuminnanoparticles are expressly included as synthetic nanocarriers.Synthetic nanocarriers according to the invention do not provoke asubstantial vector effect; preferably they do not provoke a vectoreffect. In certain preferable embodiments, synthetic nanocarriers aremodified to reduce or eliminate vector effects. This may beaccomplished, for example, by coupling various materials (e.g.polyethylene glycols) to the synthetic nanocarrier to reduce theimmunogenic nature of the synthetic nanocarrier.

A synthetic nanocarrier can be, but is not limited to, one or aplurality of lipid-based nanoparticles, polymeric nanoparticles,metallic nanoparticles, surfactant-based emulsions, dendrimers,buckyballs, nanowires, virus-like particles, peptide or protein-basedparticles (such as albumin nanoparticles) and/or nanoparticles that aredeveloped using a combination of nanomaterials such as lipid-polymernanoparticles. Synthetic nanocarriers may be a variety of differentshapes, including but not limited to spheroidal, cubic, pyramidal,oblong, cylindrical, toroidal, and the like. Synthetic nanocarriersaccording to the invention comprise one or more surfaces. Exemplarysynthetic nanocarriers that can be adapted for use in the practice ofthe present invention comprise: (1) the biodegradable nanoparticlesdisclosed in U.S. Pat. No. 5,543,158 to Gref et al., (2) the polymericnanoparticles of Published US Patent Application 20060002852 to Saltzmanet al., or (4) the lithographically constructed nanoparticles ofPublished US Patent Application 20090028910 to DeSimone et al. Syntheticnanocarriers according to the invention that have a minimum dimension ofequal to or less than about 100 nm, preferably equal to or less thanabout 100 nm, do not comprise a surface with hydroxyl groups thatactivate complement or alternatively comprise a surface that consistsessentially of moieties that are not hydroxyl groups that activatecomplement. In a preferred embodiment, synthetic nanocarriers accordingto the invention that have a minimum dimension of equal to or less thanabout 100 nm, preferably equal to or less than about 100 nm, do notcomprise a surface that substantially activates complement oralternatively comprise a surface that consists essentially of moietiesthat do not substantially activate complement. In a more preferredembodiment, synthetic nanocarriers according to the invention that havea minimum dimension of equal to or less than about 100 nm, preferablyequal to or less than about 100 nm, do not comprise a surface thatactivates complement or alternatively comprise a surface that consistsessentially of moieties that do not activate complement.

The terms “nanocarrier” “synthetic nanocarrier,” and variations thereofare generally used interchangeably herein.

T cell antigen: As used herein, the term “T cell antigen” refers to anyantigen that is recognized by and triggers an immune response in a Tcell (e.g., an antigen that is specifically recognized by a T cellreceptor on a T cell via presentation of the antigen or portion thereofbound to a major histocompatibility complex molecule (MHC). In someembodiments, an antigen that is a T cell antigen is also a B cellantigen. In other embodiments, the T cell antigen is not also a B cellantigen. T cells antigens generally are proteins or peptides. T cellantigens may be an antigen that stimulates a CD8+ T cell response, aCD4+ T cell response, or both. The nanocarriers, therefore, in someembodiments can effectively stimulate both types of responses.

Target: As used herein, the term “target” or “marker” refers to anyentity that is capable of specifically binding to a particular targetingmoiety. In some embodiments, targets are specifically associated withone or more particular tissue types. In some embodiments, targets arespecifically associated with one or more particular cell types. Forexample, a cell type specific marker is typically expressed at levels atleast 2 fold greater in that cell type than in a reference population ofcells. In some embodiments, the cell type specific marker is present atlevels at least 3 fold, at least 4 fold, at least 5 fold, at least 6fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10fold, at least 50 fold, at least 100 fold, or at least 1000 fold greaterthan its average expression in a reference population. Detection ormeasurement of a cell type specific marker may make it possible todistinguish the cell type or types of interest from cells of many, most,or all other types. In some embodiments, a target can comprise aprotein, a carbohydrate, a lipid, and/or a nucleic acid, as describedherein.

Targeted: A substance is considered to be “targeted” for the purposesdescribed herein if it specifically binds to a target. In someembodiments, a targeting moiety specifically binds to a target understringent conditions. An inventive nanocarrier, such as a vaccinenanocarrier, comprising a targeting moiety is considered to be“targeted” if the targeting moiety specifically binds to a target,thereby delivering the entire nanocarrier to a specific organ, tissue,cell, and/or subcellular locale.

Targeting moiety: As used herein, the terms “targeting moiety” and “highaffinity targeting moiety” are used interchangeably and refer to anymoiety that binds to a component of a cell. Typically, the binding of atargeting moiety to a component of a cell will be a high affinitybinding interaction. In addition to the plurality of moieties that arepresent on the immunofeature surface and providing targeting (and highavidity binding) to APCs, the nanocarriers of the invention may furthercomprise one or more additional targeting moieties. In some embodiments,the targeting moiety specifically binds to a component of a cell. Such acomponent is referred to as a “target” or a “marker.” A targeting moietymay be a polypeptide, glycoprotein, nucleic acid, small molecule,carbohydrate, lipid, etc. In some embodiments, a targeting moiety is anantibody or characteristic portion thereof. In some embodiments, atargeting moiety is a receptor or characteristic portion thereof. Insome embodiments, a targeting moiety is a ligand or characteristicportion thereof. In some embodiments, a targeting moiety is a nucleicacid targeting moiety (e.g., an aptamer) that binds to a cell typespecific marker. In some embodiments, a targeting moiety is a smallmolecule. The targeting moiety in some embodiments is on the surface ofthe nanocarrier. In other embodiments, the targeting moiety isencapsulated within the nanocarrier. In still other embodiments, thetargeting moiety is associated with the nanocarrier. In someembodiments, the targeting moiety is covalently associated with thenanocarrier. In other embodiments, the targeting moiety isnon-covalently associated with the nanocarrier. In yet otherembodiments, the targeting moiety binds a receptor expressed on thesurface of a cell. The targeting moiety, in some embodiments, binds asoluble receptor. In some embodiments, the soluble receptor is acomplement protein or a pre-existing antibody. In further embodiments,the targeting moiety is for delivery of the nanocarrier to antigenpresenting cells, T cells, or B cells. In some embodiments, the antigenpresenting cells are macrophages. In other embodiments, the macrophagesare subcapsular sinus macrophages. In still other embodiments, theantigen presenting cells are dendritic cells. In some embodiments, theantigen presenting cells are follicular dendritic cells. Specificnon-limiting examples of targeting moieties include molecules that bindto CD11b, CD169, mannose receptor, DEC-205, CD11c, CD21/CD35, CX3CR1, ora Fc receptor. In some embodiments, the molecule that binds any of theforegoing is an antibody or antigen-binding fragment thereof (e.g., ananti-CD169 antibody). In some embodiments, the molecule that binds a Fcreceptor is one that comprises the Fc portion of an immunoglobulin(e.g., IgG). In other embodiments, the Fc portion of an immunoglobulinis a human Fc portion. In some embodiments, the molecule that bindsCX3CR1 is CX3CL1 (fractalkine). Targeting moieties that bind CD169include anti-CD169 antibodies and ligands of CD169, e.g., sialylatedCD227, CD43, CD206, or portions of these ligands that retain bindingfunction, e.g., soluble portions.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” means an amount of a therapeutic,prophylactic, and/or diagnostic agent (e.g., inventive vaccinenanocarrier) that is sufficient, when administered to a subjectsuffering from or susceptible to a disease, disorder, and/or condition,to treat, alleviate, ameliorate, relieve, alleviate symptoms of,prevent, delay onset of, inhibit progression of, reduce severity of,and/or reduce incidence of the disease, disorder, and/or condition. Theterm is also intended to refer to an amount of nanocarrier orcomposition thereof provided herein that modulates an immune response ina subject.

Therapeutic agent: As used herein, the term “therapeutic agent” refersto any agent that, when administered to a subject, has a therapeutic,prophylactic, and/or diagnostic effect and/or elicits a desiredbiological and/or pharmacological effect.

Treating: As used herein, the term “treating” refers to partially orcompletely alleviating, ameliorating, relieving, delaying onset of,inhibiting progression of, reducing severity of, and/or reducingincidence of one or more symptoms or features of a particular disease,disorder, and/or condition. For example, “treating” a microbialinfection may refer to inhibiting survival, growth, and/or spread of themicrobe. Treatment may be administered to a subject who does not exhibitsigns of a disease, disorder, and/or condition and/or to a subject whoexhibits only early signs of a disease, disorder, and/or condition forthe purpose of decreasing the risk of developing pathology associatedwith the disease, disorder, and/or condition. In some embodiments,treatment comprises delivery of an inventive vaccine nanocarrier to asubject.

Universal T cell antigen: As used herein, the term “universal T cellantigen” refers to a T cell antigen that can promote T cell help andenhance an immune response to a completely unrelated antigen. UniversalT cell antigens include tetanus toxoid, as well as one or more peptidesderived from tetanus toxoid, Epstein-Barr virus, or influenza virus.Universal T cell antigens also include a component of influenza virus,such as hemagglutinin, neuraminidase, or nuclear protein, or one or morepeptides derived therefrom.

Vaccine Nanocarrier: As used herein, the term “vaccine nanocarrier”refers to a synthetic nanocarrier comprising at least oneimmunomodulatory agent or immunostimulatory agent. In certainembodiments, a vaccine nanocarrier includes at least two types ofimmunomodulatory agents. In some embodiments, the immunomodulatoryagents are antigens, and the vaccine nanocarrier comprises 2, 3, 4, 5,6, 7, 8, 9, 10, or more antigens. The different antigens can be or bederived from completely different antigenic molecules, or the differentantigens can be different epitopes from the same antigenic molecule. Inother embodiments, the vaccine nanocarrier comprises 2, 3, 4, 5, 6, 7,8, 9, 10, or more different epitopes from the same antigenic molecule. Avaccine nanocarrier may be any form of particle. A vaccine nanocarrier,in some embodiments, is capable of stimulating an immune response in Tcells and/or B cells. In other embodiments, the vaccine nanocarrier iscapable of enhancing, suppressing, directing, or redirecting an immuneresponse. In some embodiments, any assay available in the art may beused to determine whether T cells and/or B cells have been stimulated.In some embodiments, T cell stimulation may be assayed by monitoringantigen-induced production of cytokines, antigen-induced proliferationof T cells, and/or antigen-induced changes in protein expression. Insome embodiments, B cell stimulation may be assayed by monitoringantibody titers, antibody affinities, antibody performance inneutralization assays, class-switch recombination, affinity maturationof antigen-specific antibodies, development of memory B cells,development of long-lived plasma cells that can produce large amounts ofhigh-affinity antibodies for extended periods of time, germinal centerreactions, and/or antibody performance in neutralization assays. In someembodiments, a vaccine nanocarrier further comprises at least onetargeting moiety that can help deliver the vaccine nanocarrier to aparticular target (e.g., organ, tissue, cell, and/or subcellular locale)within a subject. In some embodiments, a vaccine nanocarrier furthercomprises at least one immunostimulatory agent that can help stimulatean immune response in T cells and/or B cells. In some embodiments, avaccine nanocarrier further comprises at least one nanoparticle thatallows for tunable membrane rigidity and controllable liposomestability. In some embodiments, vaccine nanocarriers comprise lipids,amphiphilic compounds, polymers, sugars, polymeric matrices, and/ornon-polymeric particles.

Vector effect: the establishment of an immune response to a syntheticnanocarrier, rather than to an antigen to which an adaptive immuneresponse is desired. Vector effects can occur when the material of thesynthetic nanocarrier is capable of stimulating a strong immune responsebecause of its chemical composition or structure.

Water soluble, non-adhesive polymer: As used herein, the term “watersoluble, non-adhesive polymer” refers to a polymer that is soluble inwater and that can confer reduced biofouling properties. In someembodiments, the water soluble, non-adhesive polymer is polyethyleneglycol, polyethylene oxide, polyalkylene glycol, and polyalkylene oxide.

Vaccines

Vaccinations are typically either passive or active in nature. Ingeneral, active vaccinations involve the exposure of a subject's immunesystem to one or more agents that are recognized as unwanted, undesired,and/or foreign and elicit an endogenous immune response resulting in theactivation of antigen-specific naive lymphocytes that then give rise toantibody-secreting B cells or antigen-specific effector and memory Tcells or both. This approach can result in long-lived protectiveimmunity that may be boosted from time to time by renewed exposure tothe same antigenic material. The prospect of longevity of a successfulimmune response to active vaccination makes this strategy more desirablein most clinical settings than passive vaccination whereby a recipientis injected with preformed antibodies or with antigen-specific effectorlymphocytes, which may confer rapid ad hoc protection, but typically donot establish persistent immunity.

A large variety of vaccine formulations are being or have been employedin humans. The most common route of administration in humans is byintramuscular (i.m.) injection, but vaccines may also be applied oradministered orally, intranasally, subcutaneously, inhalationly, orintravenously. In most cases, vaccine-derived antigens are initiallypresented to naive lymphocytes in regional lymph nodes.

Some current vaccines against, e.g., microbial pathogens, consist oflive attenuated or non-virulent variant strains of microorganisms, orkilled or otherwise inactivated organisms. Other vaccines utilize moreor less purified components of pathogen lysates, such as surfacecarbohydrates or recombinant pathogen-derived proteins that aresometimes fused to other molecules, particularly proteins that canconfer adjuvant activity.

Vaccines that utilize live attenuated or inactivated pathogens typicallyyield a vigorous immune response, but their use has limitations. Forexample, live vaccine strains can sometimes cause infectiouspathologies, especially when administered to immune-compromisedrecipients. Moreover, many pathogens, particularly viruses, undergocontinuous rapid mutations in their genome, which allow them to escapeimmune responses to antigenically distinct vaccine strains. However,most or all pathogens are thought to possess certain antigenicdeterminants that are not easily mutated because they are associatedwith essential functions. Antibodies directed against these conservedepitopes, rather than more variable, non-essential epitopes can protectagainst highly mutable viruses (Baba et al., 2000, Nat. Med., 6:200;incorporated herein by reference). Vaccines based on live or killedintact pathogens do not necessarily promote the recognition of thesecritical epitopes, but may essentially “distract” the immune system tofocus its assault on highly variable determinants Thus, the presentinvention encompasses the recognition that an engineered vaccinenanocarrier that mimics the highly immunogenic particulate nature ofviral particles, but presents selectively essential, immutable epitopes,could yield much more potent and “escape-proof” neutralizing antibodyand effector T cell responses than intact microorganisms.

The precise mechanisms by which vaccines stimulate antibody responses indraining lymph nodes (or fail to do so) are still incompletelyunderstood. B and T cells are initially sequestered in distinct anatomicregions, the superficially located B follicles and the surroundingparacortex and deep cortex, respectively. Upon antigen challenge,antigen-specific B cells in follicles as well as CD4 T cells in the Tcell area become activated and then migrate toward the border zonebetween the two compartments. B cells that have phagocytosed lymph-borneantigens process the acquired material and begin to present antigenicpeptides in MHC class-II surface molecules that are then recognized bythe activated CD4 T cells (the T_(FH) cells). Antigen-recognition allowsthe T_(FH) cells to provide help to B cells, which constitutes a potentsurvival signal and triggers the formation of germinal centers (GCs)within B follicles. The GC reaction promotes class-switch recombination,affinity maturation of antigen-specific antibodies, and the formation ofmemory B cells and long-lived plasma cells that can produce largeamounts of high-affinity antibodies for extended periods of time. Thus,the present invention encompasses the recognition that a vaccinenanocarrier may have components that allow antigenic material to beefficiently recognized by both B and T cells and to induce vigorous GCreactions (FIG. 1).

The present invention describes systems for developing vaccinenanocarriers for vaccine delivery that can overcome these aforementionedlimitations of current vaccine technology. The present inventionencompasses the recognition that lymph-borne viral particles thatmeasure tens to hundreds of nanometers in diameter and induce potentcellular and antibody responses are captured and retained on the surfaceof macrophages in the subcapsular sinus of draining lymph nodes (i.e.,subcapsular sinus macrophages, abbreviated SCS-Mph). These macrophagesare involved in the efficient early presentation of intact viralparticles to follicular B cells. In some embodiments, inventivenanocarriers mimic viral particles and target SCS-Mph. As shown inExample 1, upon subcutaneous injection of Cy5 encapsulatedpoly(lactic-coglycolic acid) (PLGA) nanoparticles (50 nm-150 nm) thatare surface stabilized with a monolayer of lipid and polyethyleneglycol, the injected nanoparticles readily enter lymphatics and arebound in the subcapsular sinus of draining lymph nodes similar tolymph-borne viruses. Similar nanocarriers carrying immunomodulatoryagent(s) that stimulate B cells and/or T cells are particularly usefulin vaccinating a subject.

Thus, the present invention encompasses the recognition thatnanocarriers, such as lymph-borne virus-sized nanocarriers carrying animmunomodulatory agent can be recognized in lymph nodes as if they wereviruses and may elicit a potent immune response, for example, when theparticles include immunomodulatory agent(s) that are recognized by Bcells and/or T cells.

By carrying immunomodulatory agents on the surface and/or loadingsimilar or distinct immunomodulatory agents inside, nanocarriers cansimultaneously deliver these immunomodulatory agents to distinct cellsof the immune system and stimulate them. In certain embodiments,immunomodulatory agents presented on nanocarrier surfaces stimulate Bcells, and immunomodulatory agents encapsulated within the nanocarriersare processed by antigen-presenting cells (APCs), such as dendriticcells (DCs), in lymphoid tissues (and by B cells after activation) andpresented to T cells. In some embodiments, by modifying the surface ofnanocarriers with a targeting moiety (e.g., antibody or fragmentthereof, peptide or polypeptide, Affibody®, Nanobody™, AdNectin™,Avimer™, aptamer, Spiegelmer®, small molecule, lipid, carbohydrate,etc.), nanocarriers can selectively deliver immunomodulatory agents tospecific antigen presenting cells, such as DCs, SCS-Mph, FDCs, T Cells,B cells, and/or combinations thereof. A nanocarrier can be, but is notlimited to, one or a plurality of lipid nanoparticles, polymericnanoparticles, metallic nanoparticles, surfactant-based emulsions,dendrimers, and/or nanoparticles that are developed using a combinationof nanomaterials such as lipid-polymer nanoparticles. Vaccinenanocarriers are described in further detail in the section entitled“Vaccine Nanocarriers.”

T Cells

The present invention provides vaccine nanocarriers for delivery of, forexample, immunomodulatory agents to the cells of the immune system. Insome embodiments, vaccine nanocarriers comprise at least oneimmunomodulatory agent which can be delivered to APCs, which thenprocess and deliver the immunomodulatory agent(s) to T cells.

Professional APCs are very efficient at internalizing antigen, either byphagocytosis or by endocytosis, and then display a fragment of theantigen, bound to either a class II major histocompatibility complex(class II MHC) molecule or a class I MHC molecule on the APC membrane.CD4 T cells recognize and interact with the antigen-class II MHCmolecule complex on the APC membrane, whereas CD8 T cells recognize andinteract with the antigen-class I MHC molecule complex. An additionalco-stimulatory signal as well as modulating cytokines are then producedby the APC, leading to T cell activation.

Immunomodulatory Agents

The present invention provides vaccine nanocarriers comprising one ormore immunomodulatory agents. In some embodiments, inventivenanocarriers comprising one or more immunomodulatory agents are used asvaccines. In some embodiments, an immunomodulatory agent may compriseisolated and/or recombinant proteins or peptides, carbohydrates,glycoproteins, glycopeptides, proteoglycans, inactivated organisms andviruses, dead organisms and virus, genetically altered organisms orviruses, and cell extracts. In some embodiments, an immunomodulatoryagent may comprise nucleic acids, carbohydrates, lipids, and/or smallmolecules. In some embodiments, an immunomodulatory agent is one thatelicits an immune response. In other embodiments, an immunomodulatoryagent is a polynucleotide that encodes a protein or peptide that whenthe protein or peptide is expressed an immune response is elicited. Insome embodiments, an immunomodulatory agent is an antigen. In someembodiments, an immunomodulatory agent is a protein or peptide. In someembodiments, an immunomodulatory agent is used for vaccines.

In some embodiments, an immunomodulatory agent is any protein and/orother antigen derived from a pathogen. The pathogen may be a virus,bacterium, fungus, protozoan, parasite, etc. In some embodiments,immunomodulatory agents may include antigens of bacterial organisms suchas Borrelia species, Bacillus anthracis, Borrelia burgdorferi,Bordetella pertussis, Camphylobacter jejuni, Chlamydia species,Chlamydial psittaci, Chlamydial trachomatis, Clostridium species,Clostridium tetani, Clostridium botulinum, Clostridium perfringens,Corynebacterium diphtheriae, Coxiella species, an Enterococcus species,Erlichia species, Escherichia coli, Francisella tularensis, Haemophilusspecies, Haemophilus influenzae, Haemophilus parainfluenzae,Lactobacillus species, a Legionella species, Legionella pneumophila,Leptospirosis interrogans, Listeria species, Listeria monocytogenes,Mycobacterium species, Mycobacterium tuberculosis, Mycobacterium leprae,Mycoplasma species, Mycoplasma pneumoniae, Neisseria species, Neisseriameningitidis, Neisseria gonorrhoeae, Pneumococcus species, Pseudomonasspecies, Pseudomonas aeruginosa, Salmonella species, Salmonella typhi,Salmonella enterica, Rickettsia species, Rickettsia ricketsii,Rickettsia typhi, Shigella species, Staphylococcus species,Staphylococcus aureus, Streptococcus species, Streptococccus pnuemoniae,Streptococcus pyrogenes, Streptococcus mutans, Treponema species,Treponema pallidum, a Vibrio species, Vibrio cholerae, Yersinia pestis,and the like.

In some embodiments, immunomodulatory agents may include antigens ofviral organisms such as pox viruses, smallpox (variola), ebola virus,hepadnavirus, marburg virus, dengue fever virus, influenza A and B,parainfluenza, respiratory syncytial virus, measles (rubeola virus),human immunodeficiency virus (HIV), human papillomavirus (HPV),varicella-zoster, herpes simplex 1 and 2, cytomegalovirus, Epstein-Barrvirus, JC virus, rhabdovirus, rotavirus, rhinovirus, adenovirus,orthomyxovirus, papillomavirus, parvovirus, picornavirus, poliovirus,mumps, rabies, reovirus, rubella, togavirus, retrovirus,coxsackieviruses, equine encephalitis, Japanese encephalitis, yellowfever, Rift Valley fever, hepatitis A, B, C, D, and E virus, and thelike. Viral organisms include those that are dsDNA viruses, ssDNAviruses, dsRNA viruses, (+) ssRNA viruses (−) sRNA viruses, ssRNA-RTviruses, and dsDNA-RT viruses.

In some embodiments, immunomodulatory agents may include antigens offungal, protozoan, and/or parasitic organisms such as Aspergillusspecies, Candida species, Candida albicans, Candida tropicalis,Cryptococcus species, Cryptococcus neoformans, Entamoeba histolytica,Histoplasma capsulatum, Leishmania species, Nocardia asteroides,Plasmodium falciparum, Toxoplasma gondii, Trichomonas vaginalis,Toxoplasma species, Trypanosoma brucei, Schistosoma mansoni, and thelike.

In some embodiments, immunomodulatory agents may include E1 and/or E2proteins of HCV. In some embodiments, immunomodulatory agents mayinclude gp120 of HIV. In some embodiments, immunomodulatory agents mayinclude hemagglutinin and/or neuraminidase of influenza virus. In someembodiments, immunomodulatory agents may include pneumococcalpolysaccharide or family 1 and/or family 2 PspA of Streptococcuspneumoniae or capsular polysaccharides types 5 and 8 or microbialsurface components recognizing adhesive matrix molecule ofStaphylococcus aureus. In some embodiments, immunomodulatory agents mayinclude mannan of Candida albicans or cryptococcal capsularpolysaccharide of Cryptococcus neoformans. In some embodiments,immunomodulatory agents may include PfEMP1 of Plasmodium falciparum orother parasite-derived antigens expressed on plasmodium-infected redblood cells or GRA7 of Toxoplasma gondi.

Any of the antigens described herein may be in the form of whole killedorganisms, peptides, proteins, glycoproteins, glycopeptides,proteoglycans, nucleic acids that encode a protein or peptide,carbohydrates, small molecules, or combinations thereof.

In some embodiments, an immunomodulatory agent is derived from amicroorganism for which at least one vaccine already exists. In someembodiments, an immunomodulatory agent is derived from a microorganismfor which no vaccines have been developed.

In some embodiments, a vaccine nanocarrier comprises at least one typeof immunomodulatory agent. In some embodiments, all of theimmunomodulatory agents of a vaccine nanocarrier are identical to oneanother. In some embodiments, a vaccine nanocarrier comprises a numberof different immunomodulatory agents. In some embodiments, a vaccinenanocarrier comprises multiple individual immunomodulatory agents, allof which are the same. In some embodiments, a vaccine nanocarriercomprises exactly one type of immunomodulatory agent. In someembodiments, a vaccine nanocarrier comprises exactly two distinct typesof immunomodulatory agents. In some embodiments, a vaccine nanocarriercomprises greater than two distinct types of immunomodulatory agents. Insome embodiments, a vaccine nanocarrier comprises 3, 4, 5, 6, 7, 8, 9,10, or more distinct types of immunomodulatory agents.

In some embodiments, a vaccine nanocarrier comprises two types ofimmunomodulatory agents which are both derived from a single genus ofmicroorganism. In some embodiments, a vaccine nanocarrier comprises twotypes of immunomodulatory agents which are both derived from a singlegenus and species of microorganism. In some embodiments, a vaccinenanocarrier comprises two types of immunomodulatory agents which areboth derived from a single genus, species, and strain of microorganism.In some embodiments, a vaccine nanocarrier comprises two types ofimmunomodulatory agents which are both derived from a single clone of amicroorganism.

In some embodiments, a vaccine nanocarrier comprises more than two typesof immunomodulatory agents which are all derived from a single genus ofmicroorganism. In some embodiments, a vaccine nanocarrier comprises morethan two types of immunomodulatory agents which are all derived from asingle genus and species of microorganism. In some embodiments, avaccine nanocarrier comprises more than two types of immunomodulatoryagents which are all derived from a single genus, species, and strain ofmicroorganism. In some embodiments, a vaccine nanocarrier comprises morethan two types of immunomodulatory agents which are all derived from asingle clone of a microorganism.

In some embodiments, a vaccine nanocarrier comprises two or more typesof immunomodulatory agent which are all derived from a single genus ofmicroorganism. In some embodiments, a vaccine nanocarrier comprises twoor more types of immunomodulatory agent which are all derived from asingle genus and species of microorganism. In some embodiments, avaccine nanocarrier comprises two or more types of immunomodulatoryagent which are all derived from a single genus, species, and strain ofmicroorganism.

In some embodiments, a vaccine nanocarrier comprises two or more typesof immunomodulatory agents which are derived from different strains of asingle species of microorganism. In some embodiments, a vaccinenanocarrier comprises two or more types of immunomodulatory agents whichare derived from different species of the same genus of microorganism.In other embodiments, a vaccine nanocarrier comprises two or more typesof immunomodulatory agents each derived from different genera ofmicroorganism.

In some embodiments, a vaccine nanocarrier comprises a single type ofimmunomodulatory agent that stimulates an immune response in both Bcells and T cells. In some embodiments, a vaccine nanocarrier comprisestwo types of immunomodulatory agents, wherein the first immunomodulatoryagent stimulates B cells, and the second type of immunomodulatory agentstimulates T cells. In certain embodiments, one or both agents maystimulate T cells and B cells. In some embodiments, a vaccinenanocarrier comprises greater than two types of immunomodulatory agents,wherein one or more types of immunomodulatory agents stimulate B cells,and one or more types of immunomodulatory agents stimulate T cells.

In some embodiments, a vaccine nanocarrier comprises at least one typeof immunomodulatory agent that is associated with the exterior surfaceof the vaccine nanocarrier. In some embodiments, the association iscovalent. In some embodiments, the covalent association is mediated byone or more linkers. In some embodiments, the association isnon-covalent. In some embodiments, the non-covalent association ismediated by charge interactions, affinity interactions, metalcoordination, physical adsorption, host-guest interactions, hydrophobicinteractions, TT stacking interactions, hydrogen bonding interactions,van der Waals interactions, magnetic interactions, electrostaticinteractions, dipole-dipole interactions, and/or combinations thereof.For a more detailed description of how an immunomodulatory agent may beassociated with a vaccine nanocarrier, please see the section belowentitled “Production of Vaccine Nanocarriers.”

In some embodiments, a vaccine nanocarrier includes a lipid membrane(e.g., lipid bilayer, lipid monolayer, etc.). At least oneimmunomodulatory agent may be associated with the lipid membrane. Insome embodiments, at least one immunomodulatory agent is embedded withinthe lipid membrane. In some embodiments, at least one immunomodulatoryagent is embedded within the lumen of a lipid bilayer. In someembodiments, a vaccine nanocarrier comprises at least oneimmunomodulatory agent that is associated with the interior surface ofthe lipid membrane. In some embodiments, at least one immunomodulatoryagent is encapsulated within the lipid membrane of a vaccinenanocarrier. In some embodiments, at least one type of immunomodulatoryagent may be located at multiple locations of a vaccine nanocarrier. Forexample, a first type of immunomodulatory agent may be embedded within alipid membrane, and a second type of immunomodulatory agent may beencapsulated within the lipid membrane of a vaccine nanocarrier. To giveanother example, a first type of immunomodulatory agent may beassociated with the exterior surface of a lipid membrane, and a secondtype of immunomodulatory agent may be associated with the interiorsurface of the lipid membrane of a vaccine nanocarrier. In someembodiments, a first type of immunomodulatory agent may be embeddedwithin the lumen of a lipid bilayer of a vaccine nanocarrier, and thelipid bilayer may encapsulate a polymeric matrix throughout which asecond type of immunomodulatory agent is distributed. In someembodiments, a first type of immunomodulatory agent and a second type ofimmunomodulatory agent may be in the same locale of a vaccinenanocarrier (e.g., they may both be associated with the exterior surfaceof a vaccine nanocarrier; they may both be encapsulated within thevaccine nanocarrier; etc.).

In some embodiments, a vaccine nanocarrier includes a polymer (e.g., apolymeric core). At least one type of immunomodulatory agent may beassociated with the polymer. In some embodiments, at least one type ofimmunomodulatory agent is embedded within the polymer. In someembodiments, a vaccine nanocarrier comprises at least one type ofimmunomodulatory agent that is associated with the interior surface ofthe polymer. In some embodiments, at least one type of immunomodulatoryagent is encapsulated with the polymer of a vaccine nanocarrier. In someembodiments, at least one type of immunomodulatory agent may be locatedat multiple locations of a vaccine nanocarrier. For example, a firsttype of immunomodulatory agent may be embedded within a polymer, and asecond type of immunomodulatory agent may be encapsulated within a lipidmembrane surrounding the polymeric core of a vaccine nanocarrier. Togive another example, a first type of immunomodulatory agent may beassociated with the exterior surface of a polymer, and a second type ofimmunomodulatory agent may be embedded within the polymer of a vaccinenanocarrier.

One of ordinary skill in the art will recognize that the precedingexamples are only representative of the many different ways in whichmultiple immunomodulatory agents may be associated with differentlocales of vaccine nanocarriers. Multiple immunomodulatory agents may belocated at any combination of locales of vaccine nanocarriers.Additionally, the aforementioned examples can also apply to the otheragents of a nanocarrier (e.g., a immunostimulatory agent).

In some embodiments, the immunomodulatory agent is a T cell antigen, andthe T cell antigen is derived from the same pathogen against whichvaccination is intended. In this case, an initially small number ofnaive T cells are stimulated to generate pathogen-specific effector andmemory T cells. In some embodiments, the antigen may be taken from anunrelated source, such as an infectious agent to which wide-spreadimmunity already exists (e.g., tetanus toxoid or a common component ofinfluenza virus, such as hemagglutinin, neuraminidase, or nuclearprotein). In the latter case, the vaccine exploits the presence ofmemory T cells that have arisen in response to prior infections orvaccinations. Memory cells in general react more rapidly and vigorouslyto antigen rechallenge and, therefore, may provide a superior source ofhelp to B cells.

Other T cell antigens include, but are not limited to, degenerativedisease antigens, infectious disease antigens, cancer antigens,alloantigens, atopic disease antigens, autoimmune disease antigens,contact sensitizers, haptens, xenoantigens, or metabolic disease enzymesor enzymatic products thereof. In some embodiments, the infectiousdisease antigen is a viral antigen, which includes any antigen derivedfrom any of the viruses described herein. Examples of T cell antigensinclude those provided elsewhere herein.

In some embodiments, T cell antigens are incorporated into nanocarriersas intact proteins. In some embodiments, T cell antigens areincorporated into nanocarriers as modified proteins. In someembodiments, T cell antigens are incorporated into nanocarriers asmutated proteins. In some embodiments, T cell antigens are provided as acollection of overlapping peptides, which can boost antigenincorporation into MHC class II complexes and, therefore, furtherpromote a helper response. In some embodiments, T cell antigens areprovided as a collection of non-overlapping peptides, which can boostantigen incorporation into MHC class II complexes and, therefore,further promote a helper response. In some embodiments, T cell antigensare provided as nucleic acids that encode the antigens.

In some embodiments, inventive nanocarriers, such as vaccinenanocarriers, comprise less than less than 90% by weight, less than 75%by weight, less than 50% by weight, less than 40% by weight, less than30% by weight, less than 20% by weight, less than 15% by weight, lessthan 10% by weight, less than 5% by weight, less than 1% by weight, orless than 0.5% by weight of the immunomodulatory agent.

Targeting Moieties

In some embodiments, inventive nanocarriers comprise one or moretargeting moieties. In certain embodiments of the invention,nanocarriers are associated with one or more targeting moieties. Atargeting moiety is any moiety that binds to a component associated withan organ, tissue, cell, extracellular matrix, and/or subcellular locale.In some embodiments, such a component is referred to as a “target” or a“marker,” and these are discussed in further detail below.

A targeting moiety may be a nucleic acid, polypeptide, glycoprotein,carbohydrate, lipid, small molecule, etc. For example, a targetingmoiety can be a nucleic acid targeting moiety (e.g. an aptamer,Spiegelmer®, etc.) that binds to a cell type specific marker. Ingeneral, an aptamer is an oligonucleotide (e.g., DNA, RNA, or an analogor derivative thereof) that binds to a particular target, such as apolypeptide. In some embodiments, a targeting moiety may be a naturallyoccurring or synthetic ligand for a cell surface receptor, e.g., agrowth factor, hormone, LDL, transferrin, etc. A targeting moiety can bean antibody, which term is intended to include antibody fragments,characteristic portions of antibodies, single chain antibodies, etc.Synthetic binding proteins such as Affibodies®, Nanobodies™, AdNectins™,Avimers™, etc., can be used. Peptide targeting moieties can beidentified, e.g., using procedures such as phage display. This widelyused technique has been used to identify cell specific ligands for avariety of different cell types.

In accordance with the present invention, a targeting moiety recognizesone or more “targets” or “markers” associated with a particular organ,tissue, cell, and/or subcellular locale. In some embodiments, a targetmay be a marker that is exclusively or primarily associated with one ora few cell types, with one or a few diseases, and/or with one or a fewdevelopmental stages. A cell type specific marker is typically expressedat levels at least 2 fold greater in that cell type than in a referencepopulation of cells which may consist, for example, of a mixturecontaining an approximately equal amount of cells (e.g., approximatelyequal numbers of cells, approximately equal volume of cells,approximately equal mass of cells, etc.). In some embodiments, the celltype specific marker is present at levels at least 3 fold, at least 4fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100fold, at least 500 fold, at least 1000 fold, at least 5000 fold, or atleast 10,000 fold greater than its average expression in a referencepopulation. Detection or measurement of a cell type specific marker maymake it possible to distinguish the cell type or types of interest fromcells of many, most, or all other types.

In some embodiments, a target can comprise a protein, a carbohydrate, alipid, and/or a nucleic acid. In certain embodiments, a target cancomprise a protein and/or characteristic portion thereof, such as atumor-marker, integrin, cell surface receptor, transmembrane protein,intercellular protein, ion channel, membrane transporter protein,enzyme, antibody, chimeric protein, glycoprotein, etc. In certainembodiments, a target can comprise a carbohydrate and/or characteristicportion thereof, such as a glycoprotein, sugar (e.g., monosaccharide,disaccharide, polysaccharide), glycocalyx (i.e., the carbohydrate-richperipheral zone on the outside surface of most eukaryotic cells) etc. Incertain embodiments, a target can comprise a lipid and/or characteristicportion thereof, such as an oil, fatty acid, glyceride, hormone, steroid(e.g., cholesterol, bile acid), vitamin (e.g. vitamin E), phospholipid,sphingolipid, lipoprotein, etc. In certain embodiments, a target cancomprise a nucleic acid and/or characteristic portion thereof, such as aDNA nucleic acid; RNA nucleic acid; modified DNA nucleic acid; modifiedRNA nucleic acid; nucleic acid that includes any combination of DNA,RNA, modified DNA, and modified RNA; etc.

In some embodiments, a targeting moiety could be the surfaceglycoprotein molecule from VSV. VSV comprises a single surface molecule,VSV-G, which is a toll-like receptor agonist. VSV is efficientlytargeted to cells of the immune system, so in some embodiments, vaccinenanocarriers could comprise the VSV surface molecule in order to targetvaccine nanocarriers to cells of the immune system.

In some embodiments, a target is a tumor marker. In some embodiments, atumor marker is an antigen that is expressed in tumor cells but not inhealthy and/or normal cells. In some embodiments, a tumor marker is anantigen that is more prevalent in tumor cells than in healthy and/ornormal cells. Exemplary tumor markers include, but are not limited to,gp100; Melan-A; tyrosinase; PSMA; HER-2/neu; MUC-1; topoisomerase IIα;sialyl-Tn; carcinoembryonic antigen; ErbB-3-binding protein-1;alpha-fetoprotein; and the cancer-testis antigens MAGE-A1, MAGE A4, andNY-ESO-1.

In some embodiments, a target is an APC marker. In some embodiments, anAPC target is an antigen that is expressed in APCs but not in non-APCs.In some embodiments, an APC target is an antigen that is more prevalentin APCs than in non-APCs. Exemplary APC markers include, but are notlimited to, CD11c, CD11b, CD14, CD40, CD45, CD163, CD169 (sialoadhesin),DEC205 (CD205), MHC class II, DC-SIGN, CD21/CD35, and FcγRI, PD-L2. Insome embodiments, APC markers include any of DC and/or macrophagemarkers, examples of which are described herein.

In certain embodiments, a target is a DC marker. In some embodiments, aDC target is an antigen that is expressed in DCs but not in non-DCs. Insome embodiments, a DC target is an antigen that is more prevalent inDCs than in non-DCs. Exemplary DC markers are listed below in thesection entitled “Dendritic Cells” and include those provided elsewhereherein.

In certain embodiments, a target is a T cell marker. In someembodiments, a T cell target is an antigen that is expressed in T cellsbut not in non-T cells. In some embodiments, a T cell target is anantigen that is more prevalent in T cells than in non-T cells. ExemplaryT cell markers are listed below in the section entitled “T CellTargeting Moieties” and include those provided elsewhere herein.

In some embodiments, a target is preferentially expressed in particularcell types. For example, expression of an APC, DC, and/or T cell targetin APCs, DCs, and/or T cells is at least 2-fold, at least 3-fold, atleast 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, atleast 50-fold, at least 100-fold, at least 500-fold, or at least1000-fold overexpressed in APCs, DCs, and/or T cells relative to areference population. In some embodiments, a reference population maycomprise non-APCs, FDCs, and/or T cells.

In some embodiments, expression of an APC, DC, and/or T cell target inactivated APCs, DCs, and/or T cells is at least 2-fold, at least 3-fold,at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, atleast 50-fold, at least 100-fold, at least 500-fold, or at least1000-fold overexpressed in activated APCs, DCs, and/or T cells relativeto a reference population. In some embodiments, a reference populationmay comprise non-activated APCs, DCs, and/or T cells.

In some embodiments, inventive nanocarriers, such as vaccinenanocarriers, comprise less than 50% by weight, less than 40% by weight,less than 30% by weight, less than 20% by weight, less than 15% byweight, less than 10% by weight, less than 5% by weight, less than 1% byweight, or less than 0.5% by weight of the targeting moiety.

In some embodiments, targeting moieties are covalently associated with ananocarrier. In some embodiments, covalent association is mediated by alinker. In some embodiments, targeting moieties are not covalentlyassociated with a nanocarrier. For example, targeting moieties may beassociated with the surface of, encapsulated within, surrounded by,and/or distributed throughout the polymeric matrix of an inventiveparticle. For example, in some embodiments, a targeting moiety can beencapsulated within, surrounded by, and/or dispersed throughout theliposomal membrane and/or polymeric matrix of a nanocarrier.Alternatively or additionally, a targeting moiety can be associated witha nanocarrier by charge interactions, affinity interactions, metalcoordination, physical adsorption, host-guest interactions, hydrophobicinteractions, TT stacking interactions, hydrogen bonding interactions,van der Waals interactions, magnetic interactions, electrostaticinteractions, dipole-dipole interactions, and/or combinations thereof.Association of targeting moieties with vaccine nanocarriers is describedin further detail below, in the section entitled “Production of VaccineNanocarriers.”

Dendritic Cells

Dendritic Cells (DCs) are a type of myeloid leukocytes; they are amongthe most potent antigen presenting cells for T lymphocytes. Resting DCsreside in many tissues, including lymph nodes, in an immature,tolerogenic state, i.e., they present intermediate to high levels ofpeptide-MHC complexes, but with little or no costimulatory molecules andwithout secreting cytokines that T cells need to differentiate intoeffector cells. T cells that are presented with a specific antigen byimmature DCs begin to proliferate for a few days, but then they die byapoptosis or become unresponsive to further activation. The ensuingdepletion of antigen-specific T cell responses renders the hostselectively tolerant to this antigen. By contrast, when DCs acquireantigens while they are exposed to maturation stimuli, the cells rapidlyup-regulate MHC and costimulatory molecules and secrete severalcytokines. The now mature DCs are potent inducers of effector T cellsand immunological memory. DC maturation can be induced by many signals,such as certain inflammatory cytokines, ligation of DC-expressed CD40,agonists for TLRs, (e.g., bacterial endotoxin), immune complexes,activated complement, necrotic cells, apoptotic cells, free urate, uratecrystals, and/or HMGB-1.

DEC-205 (i.e., CD205) is a surface-expressed multi-functional lectinthat is selectively expressed on DCs and thymic epithelial cells inlymphoid tissues. In vivo experiments with subcutaneously injectedchimeric α-DEC-205 monoclonal antibodies have shown that ligand bindingto DEC-205 induces efficient internalization and subsequent processingof the endocytosed material for presentation in MHC molecules in bothmice and humans (Hawiger et al., 2001, J. Exp. Med. 194:769; Bonifaz etal., 2002, J. Exp. Med., 196:1627; and Bozzacco et al., 2007, Proc.Natl. Acad. Sci., USA, 104:1289; each of which is incorporated herein byreference). Upon intra-cutaneous or subcutaneous injection, the chimericantibody is transported via lymph vessels to the draining lymph nodeswhere it binds specifically to resident DCs, thus providing the means totarget antigens to resting DCs without causing their maturation. Thetargeted DCs will then induce T cell tolerance to the presented antigen,rather than immunity. However, when DEC-205 is targeted together with animmunostimulatory agent that induces DC maturation (e.g., α-CD40 or oneor more ligands for DC-expressed TLRs; discussed in further detail belowin the section entitled “Immunostimulatory Agents”), then the vaccineacts as a potent immunostimulant promoting preferentially cytotoxic andTh1-type effector T cell responses.

DC targeting can be accomplished by moieties that bind DC-205, CD11c,class II MHC, CD80, CD86, DC-SIGN, CD11b, BDCA-1, BDCA-2, BDCA-4,Siglec-H, CX3CR1, and/or Langerin.

In some embodiments, DC targeting can be accomplished by any targetingmoiety that specifically binds to any entity (e.g., protein, lipid,carbohydrate, small molecule, etc.) that is prominently expressed and/orpresent on DCs (i.e., a DC marker). Exemplary DC markers include, butare not limited to, CD1a (R4, T6, HTA-1); CD1b (R1); CD1c (M241, R7);CD1d (R3); CD1e (R2); CD11b (αM Integrin chain, CR3, Mo1, C3niR, Mac-1);CD11c (αX Integrin, p150, 95, AXb2); CDw117 (Lactosylceramide, LacCer);CD19 (B4); CD33 (gp67); CD 35 (CR1, C3b/C4b receptor); CD 36 (GpIIIb,GPIV, PASIV); CD39 (ATPdehydrogenase, NTPdehydrogenase-1); CD40 (Bp50);CD45 (LCA, T200, B220, Ly5); CD45RA; CD45RB; CD45RC; CD45RO (UCHL-1);CD49d (VLA-4α, α4 Integrin); CD49e (VLA-5α, α5 Integrin); CD58 (LFA-3);CD64 (FcγRI); CD72 (Ly-19.2, Ly-32.2, Lyb-2); CD73 (Ecto-5′nucloticlase); CD74 (Ii, invariant chain); CD80 (B7, B7-1, BB1); CD81(TAPA-1); CD83 (HB15); CD85a (ILT5, LIR3, HL9); CD85d (ILT4, LIR2,MIR10); CD85j (ILT2, LIR1, MIR7); CD85k (ILT3, LIR5, HM18); CD86(B7-2/B70); CD88 (C5aB); CD97 (BL-KDD/F12); CD101 (IGSF2, P126, V7);CD116 (GM-CSFRα); CD120a (TMFRI, p55); CD120b (TNFRII, p75, TNFR p80);CD123 (IL-3Rα); CD139; CD148 (HPTP-η, p260, DEP-1); CD150 (SLAM, IPO-3);CD156b (TACE, ADAM17, cSVP); CD157 (Mo5, BST-1); CD167a (DDR1, trkE,cak); CD168 (RHAMM, 1HABP, HMMR); CD169 (Sialoadhesin, Siglec-1); CD170(Siglec-5); CD171 (L1CAM, NILE); CD172 (SIRP-1α, MyD-1); CD172b (SIRPβ);CD180 (RP105, Bgp95, Ly64); CD184 (CXCR4, NPY3R); CD193 (CCR3); CD196(CCR6); CD197 (CCR7 (ws CDw197)); CDw197 (CCR7, EBI1, BLR2); CD200(OX2); CD205 (DEC-205); CD206 (MMR); CD207 (Langerin); CD208 (DC-LAMP);CD209 (DC-SIGN); CDw218a (IL18Rα); CDw218b (IL8Rβ); CD227 (MUC1, PUM,PEM, EMA); CD230 (Prion Protein (PrP)); CD252 (OX40L, TNF (ligand)superfamily, member 4); CD258 (LIGHT, TNF (ligand) superfamily, member14); CD265 (TRANCE-R, TNF-R superfamily, member 11a); CD271 (NGFR, p75,TNFR superfamily, member 16); CD273 (B7DC, PDL2); CD274 (B7H1, PDL1);CD275 (B7H2, ICOSL); CD276 (B7H3); CD277 (BT3.1, B7 family: Butyrophilin3); CD283 (TLR3, TOLL-like receptor 3); CD289 (TLR9, TOLL-like receptor9); CD295 (LEPR); CD298 (ATP1B3, Na K ATPase β3 submit); CD300a(CMRF-35H); CD300c (CMRF-35A); CD301 (MGL1, CLECSF14); CD302 (DCL1);CD303 (BDCA2); CD304 (BDCA4); CD312 (EMR2); CD317 (BST2); CD319 (CRACC,SLAMF7); CD320 (8D6); and CD68 (gp110, Macrosialin); class II MHC;BDCA-1; Siglec-H; wherein the names listed in parentheses representalternative names.

T Cell Targeting Moieties

In some embodiments, T cell targeting can be accomplished by anytargeting moiety that specifically binds to any entity (e.g., protein,lipid, carbohydrate, small molecule, etc.) that is prominently expressedand/or present on T cells (i.e., a T cell marker). Exemplary T cellmarkers include, but are not limited to, CD2 (E-rosette R, T11, LFA-2);CD3 (T3); CD3α; CD3β; CD3ε; CD4 (L3T4, W3/25, T4); CD5 (T1, Tp67, Leu-1,LY-1); CD6 (T12); CD7 (gp40, Leu 9); CD8a (Leu2, T8, Lyt-2,3); CD8b(CD8, Leu2, Lyt3); CD11a (LFA-1α, α Integrin chain); CD11b (αM Integrinchain, CR3, Mo1, C3niR, Mac-1); CD11c (αX Integrin, p150, 95, AXb2);CD15s (Sialyl Lewis X); CD15u (3′ sulpho Lewis X); CD15su (6sulpho-sialyl Lewis X); CD16b (FcgRIIIb); CDw17 (Lactosylceramide,LacCer); CD18 (Integrin β2 CD11a, b, c (β-subunit); CD26 (DPP IVectoeneyme, ADA binding protein); CD27 (T14, S152); CD28 (Tp44, T44);CD29 (Platelet GPIIa, β-1 integrin, GP); CD31 (PECAM-1, Endocam); CD35(CR1, C3b/C4b receptor); CD37 (gp52-40); CD38 (ADP-ribosyl/cyclase,T10); CD43 (Sialophorin, Leukosialin); CD44 (ECMRII, H-CAM, Pgp-1); CD45(LCA, T200, B220, Ly5); CD45RA (p561ck, p59fyn, Src kinases); CD45RB(p561ck, p59fyn, Src kinases); CD45RC (p561ck, p59fyn, Src kinases);CD46 (MCP); CD47 (gp42, IAP, OA3, Neurophillin); CD47R (MEM-133); CD48(Blast-1, Hulym3, BCM-1, OX-45); CD49c (VLA-3α, α3 Integrin); CD49d(VLA-4α, α4 Integrin); CD49e (VLA-5α, α5 Integrin); CD49f (VLA-6α, α6Integrin gplc); CD50 (ICAM-3); CD52 (CAMPATH-1, HES); CD53 (OX-44); CD54(ICAM-1); CD55 (DAF); CD56 (Leu-19, NKH-1, NCAM); CD57 (HNK1, Leu-7);CD58 (LFA-3); CD59 (1F5Ag, H19, Protectin, MACIF, MIRL, P-18); CD60a(GD3); CD60b (9-O-acetyl GD3); CD60c (7-O acetyl GD3); CD62L(L-selectin, LAM-1, LECAM-1, MEL-14, Leu8, TQ1); CD73(Ecto-5′-nuclotidase); CD75 (sialo-masked Lactosamine); CD75S (α2, 6sialylated Lactosamine); CD81 (TAPA-1); CD82 (4F9, C33, IA-4, KAI1, R2);CD84 (P75, GR6); CD85a (ILT5, LIR3, HL9); CD85j (ILT2, LIR1, MIR7); CD87(uPAR); CDw92 (p70); CD94 (Kp43); CD95 (APO-1, FAS, TNFRSF6); CD98 (4F2,FRP-1, RL-388); CD99 (MIC2, E2); CD99R (CD99 Mab restricted); CD100(SEMA4D); CD102 (ICAM-2); CD108 (SEMA7A, JMH blood group antigen);CDw119 (IFNγR, IFNγRa); CD120a (TNFRI, p55); CD120b (TNFRII, p75, TNFRp80); CD121a (Type 1 IL-1R); CD121b (Type 2 IL-1R); CD122 (IL2Rβ); CD124(IL-4Rα); CD126 (IL-6Rα); CD127 (p90, IL-7R, IL-7Rα); CD128a (IL-8Ra,CXCR1, (Tentatively renamed as CD181)); CD128b (IL-8Rb, CXCR2,(Tentatively renamed as CD182)); CD130 (gp130); CD132 (Common γ chain,IL-2Rγ); CD147 (Basigin, EMMPRIN, M6, OX47); CD148 (HPTP-η, p260,DEP-1); CD150 (SLAM, IPO-3); CD153 (CD30L, TNSF8); CD156b (TACE, ADAM17,cSVP); CD158a (KIR2DL1, p58.1); CD158b1 (KIR2DL2, p58.2); CD158b2(KIR2DL3, p58.3); CD158c (KIR2DS6, KIRX); CD158|e1/e2 (KIR3DLI/S1, p70);CD159F (KIR2DL5); CD158g (KIR2DS5); CD158h (KIR2DS1, p50.1); CD158i(KIR2DS4, p50.3); CD158j (KIR2DS2, p50.2); CCD158k (KIR3DL2, p140);CD159a (NKG2A); CD160 (BY55, NK1, NK28); CD161 (NKR, NKRP1A); CD162(PSGL-1); CD164 (MGC-24, MUC-24); CD171 (L1CAM, NILE); CD172g (SIRPg);CD181 (CXCR1, (Formerly known as CD128a)); CD182 (CXCR2, (Formerly knownas CD128b)); CD183 (CXCR3, GPR9); CD184 (CXCR4, NPY3R); CD185 (CXCR5);CD186 (CXCR6); CD191 (CCR1); CD192 (CCR2); CD193 (CCR3); CD195 (CCR5);CD196 (CCR6); CD197 (CCR7 (was CDw197)); CDw197 (CCR7, EBI1, BLR2);CDw198 (CCR8); CDw199 (CCR9); CD205 (DEC-205); CDw210 (CK); CDw217 (CK);CDw218a (IL18Rα); CDw218b (IL18Rβ); CD220 (Insulin R); CD221 (IGF1 R);CD222 (M6P-R, IGFII-R); CD223 (LAG-3); CD224 (GGT); CD225 (Leu13); CD226(DNAM-1, PTA1); CD229 (Ly9); CD230 (Prion Protein (PrP)); CD244 (2B4,P38, NAIL); CD245 (p220/240); CD247 (CD3 Zeta Chain); CD261 (TRAIL-R1,TNF-R superfamily, member 10a); CD262 (TRAIL-R2, TNF-R superfamily,member 10b); CD263 (TRAIL-R3, TNF-R superfamily, member 10c); CD264(TRAIL-R4, TNF-R superfamily, member 10d); CD265 (TRANCE-R, TNF-Rsuperfamily, member 11a); CD268 (BAFFR, TNF-R superfamily, member 13C);CD272 (BTLA); CD275 (B7H2, ICOSL); CD277 (BT3.1, B7 family: Butyrophilin3); CD294 (CRTH2, PGRD2, G protein-coupled receptor 44); CD295 (LEPR);CD296 (ART1, ADP-ribosyltransferase 1); CD298 (ATP1B3, Na K ATPase β3subunit); CD300a (CMRF-35H); CD300c (CMRF-35A); CD305 (LAIR1); CD314(NKG2D); CD316 (EW12); CD317 (BST2); CD319 (CRACC, SLAMF7); CD321(JAM1); CD322 (JAM2); CDw328 (Siglec7); and CD68 (gp 110, Macrosialin);wherein the names listed in parentheses represent alternative names.

In some embodiments, T cell targeting can be accomplished by anytargeting moiety that binds, such as specifically binds, to any entity(e.g., protein, lipid, carbohydrate, small molecule, etc.) that isprominently expressed and/or present on T cells upon activation (i.e.,activated T cell targets). Exemplary activated T cell targeting moietiesinclude, but are not limited to, CD1a (RA, T6, HTA-1); CD1b (R1); Cd1c(M241,R7); CD1d (R3); CD9 (p24, DRAP-1, MRP-1); CD25 (Tac antigen,IL-2Rα, p55); CD30 (Ber-H2, Ki-1); CD39 (ATPdehydrogenase,NTPdehydrogenase-1); CD45RO (UCHL-1); CD49a (VLA-1α, α1 Integrin); CD49b(VLA-2α, gp1a, α2 Integrin); CD69 (AIM, EA 1, MLR3, gp34/28, VEA); CD70(Ki-24, CD27 ligand); CD74 (Ii, invariant chain); CD80 (B7, B7-1, BB1);CD86 (B7-2/B70); CD96 (TACTILE); CD97 (BL-KDD/F12); CD101 (IGSF2, P126,V7); CD103 (HML-1, Integrin αE, ITGAE); CD107a (LAMP-1); CD107b(LAMP-2); CD109 (8A3, E123 7D1); CD134 (OX40, TNFRSF4); CDw137 (4-1BB,ILA); CD146 (Muc 18, S-endo, MCAM, MeI-CAM); CD152 (CTLA-4); CD154(CD40L, gp39, TRAP-1, T-BAM); CD166 (ALCAM, KG-CAM, SC-1, BEN,DM-GRASP); CD178 (Fas Ligand); CD227 (MUC1, PUM, PEM, EMA); CD253(TRAIL, TNF (ligand) superfamily, member 10); CD254 (TRANCE, RANKL, TNF(ligand) superfamily, member 11); CD258 (LIGHT, TMF (ligand)superfamily, member 14); CD267 (TACI, TNF-R superfamily, member 13B);CD273 (B7DC, PDL2); CD274 (B7H1, PDL1); CD278 (ICOS); CD279 (PD1); andCD312 (EMR2); wherein the names listed in parentheses representalternative names.

Molecular Characteristics of Targeting Moieties

Nucleic Acid Targeting Moieties. As used herein, a “nucleic acidtargeting moiety” is a nucleic acid that binds selectively to a target.In some embodiments, a nucleic acid targeting moiety is a nucleic acidaptamer. An aptamer is typically a polynucleotide that binds to aspecific target structure that is associated with a particular organ,tissue, cell, extracellular matrix component, and/or subcellular locale.In general, the targeting function of the aptamer is based on thethree-dimensional structure of the aptamer. In some embodiments, bindingof an aptamer to a target is typically mediated by the interactionbetween the two- and/or three-dimensional structures of both the aptamerand the target. In some embodiments, binding of an aptamer to a targetis not solely based on the primary sequence of the aptamer, but dependson the three-dimensional structure(s) of the aptamer and/or target. Insome embodiments, aptamers bind to their targets via complementaryWatson-Crick base pairing which is interrupted by structures (e.g.,hairpin loops) that disrupt base pairing.

In some embodiments, a nucleic acid targeting moiety is a Spiegelmer®.In general, Spiegelmers® are high-affinity L-enantiomericoligonucleotide ligands that display high resistance to enzymaticdegradation compared with D-oligonucleotides. In some embodiments,Spiegelmers® can be designed and utilized just as an aptamer would bedesigned and utilized.

One of ordinary skill in the art will recognize that any nucleic acidthat is capable of specifically binding to a target, as describedherein, can be used in accordance with the present invention.

Nucleic acids of the present invention (including nucleic acid targetingmoieties and/or functional RNAs to be delivered, e.g., RNAi agents,ribozymes, tRNAs, etc., described in further detail below) may beprepared according to any available technique including, but not limitedto chemical synthesis, enzymatic synthesis, enzymatic or chemicalcleavage of a longer precursor, etc. Methods of synthesizing RNAs areknown in the art (see, e.g., Gait, M. J. (ed.) Oligonucleotidesynthesis: a practical approach, Oxford [Oxfordshire], Washington, D.C.:IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis:methods and applications, Methods in molecular biology, v. 288 (Clifton,N.J.) Totowa, N.J.: Humana Press, 2005; both of which are incorporatedherein by reference).

The nucleic acid that forms the nucleic acid targeting moiety maycomprise naturally occurring nucleosides, modified nucleosides,naturally occurring nucleosides with hydrocarbon linkers (e.g., analkylene) or a polyether linker (e.g., a PEG linker) inserted betweenone or more nucleosides, modified nucleosides with hydrocarbon or PEGlinkers inserted between one or more nucleosides, or a combination ofthereof. In some embodiments, nucleotides or modified nucleotides of thenucleic acid targeting moiety can be replaced with a hydrocarbon linkeror a polyether linker provided that the binding affinity and selectivityof the nucleic acid targeting moiety is not substantially reduced by thesubstitution (e.g., the dissociation constant of the nucleic acidtargeting moiety for the target should not be greater than about 1×10⁻³M).

It will be appreciated by those of ordinary skill in the art thatnucleic acids in accordance with the present invention may comprisenucleotides entirely of the types found in naturally occurring nucleicacids, or may instead include one or more nucleotide analogs or have astructure that otherwise differs from that of a naturally occurringnucleic acid. U.S. Pat. Nos. 6,403,779; 6,399,754; 6,225,460; 6,127,533;6,031,086; 6,005,087; 5,977,089; and references therein disclose a widevariety of specific nucleotide analogs and modifications that may beused. See Crooke, S. (ed.) Antisense Drug Technology: Principles,Strategies, and Applications (1^(st) ed), Marcel Dekker; ISBN:0824705661, 1st edition (2001); incorporated herein by reference; andreferences therein. For example, 2′-modifications include halo, alkoxyand allyloxy groups. In some embodiments, the 2′-OH group is replaced bya group selected from H, OR, R, halo, SH, SR₁, NH₂, NH_(R), NR₂ or CN,wherein R is C₁-C₆ alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br orI. Examples of modified linkages include phosphorothioate and5′-N-phosphoramidite linkages.

Nucleic acids comprising a variety of different nucleotide analogs,modified backbones, or non-naturally occurring internucleoside linkagescan be utilized in accordance with the present invention. Nucleic acidsof the present invention may include natural nucleosides (i.e.,adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine,deoxythymidine, deoxyguanosine, and deoxycytidine) or modifiednucleosides. Examples of modified nucleotides include base modifiednucleosides (e.g., aracytidine, inosine, isoguanosine, nebularine,pseudouridine, 2,6-diaminopurine, 2-aminopurine, 2-thiothymidine,3-deaza-5-azacytidine, 2′-deoxyuridine, 3-nitropyrrole, 4-methylindole,4-thiouridine, 4-thiothymidine, 2-aminoadenosine, 2-thiothymidine,2-thiouridine, 5-bromocytidine, 5-iodouridine, inosine, 6-azauridine,6-chloropurine, 7-deazaadenosine, 7-deazaguanosine, 8-azaadenosine,8-azidoadenosine, benzimidazole, M1-methyladenosine, pyrrolo-pyrimidine,2-amino-6-chloropurine, 3-methyl adenosine, 5-propynylcytidine,5-propynyluridine, 5-bromouridine, 5-fluorouridine, 5-methylcytidine,7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,O(6)-methylguanine, and 2-thiocytidine), chemically or biologicallymodified bases (e.g., methylated bases), modified sugars (e.g.,2′-fluororibose, 2′-aminoribose, 2′-azidoribose, 2′-O-methylribose,L-enantiomeric nucleosides arabinose, and hexose), modified phosphategroups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages), andcombinations thereof. Natural and modified nucleotide monomers for thechemical synthesis of nucleic acids are readily available. In somecases, nucleic acids comprising such modifications display improvedproperties relative to nucleic acids consisting only of naturallyoccurring nucleotides. In some embodiments, nucleic acid modificationsdescribed herein are utilized to reduce and/or prevent digestion bynucleases (e.g. exonucleases, endonucleases, etc.). For example, thestructure of a nucleic acid may be stabilized by including nucleotideanalogs at the 3′ end of one or both strands order to reduce digestion.

Modified nucleic acids need not be uniformly modified along the entirelength of the molecule. Different nucleotide modifications and/orbackbone structures may exist at various positions in the nucleic acid.One of ordinary skill in the art will appreciate that the nucleotideanalogs or other modification(s) may be located at any position(s) of anucleic acid such that the function of the nucleic acid is notsubstantially affected. To give but one example, modifications may belocated at any position of an aptamer such that the ability of theaptamer to specifically bind to the aptamer target is not substantiallyaffected. The modified region may be at the 5′-end and/or the 3′-end ofone or both strands. For example, modified aptamers in whichapproximately 1 to approximately 5 residues at the 5′ and/or 3′ end ofeither of both strands are nucleotide analogs and/or have a backbonemodification can be employed. The modification may be a 5′ or 3′terminal modification. A nucleic acid strand may comprise at least 50%unmodified nucleotides, at least 80% unmodified nucleotides, at least90% unmodified nucleotides, or 100% unmodified nucleotides.

Nucleic acids in accordance with the present invention may, for example,comprise a modification to a sugar, nucleoside, or internucleosidelinkage such as those described in U.S. Patent Publications2003/0175950, 2004/0192626, 2004/0092470, 2005/0020525, and2005/0032733. The present invention encompasses the use of any nucleicacid having any one or more of the modification described therein. Forexample, a number of terminal conjugates, e.g., lipids such ascholesterol, lithocholic acid, aluric acid, or long alkyl branchedchains have been reported to improve cellular uptake. Analogs andmodifications may be tested using, e.g., using any appropriate assayknown in the art, for example, to select those that result in improveddelivery of a therapeutic agent, improved specific binding of an aptamerto an aptamer target, etc. In some embodiments, nucleic acids inaccordance with the present invention may comprise one or morenon-natural nucleoside linkages. In some embodiments, one or moreinternal nucleotides at the 3′-end, 5′-end, or both 3′- and 5′-ends ofthe aptamer are inverted to yield a linkage such as a 3′-3′ linkage or a5′-5′ linkage.

In some embodiments, nucleic acids in accordance with the presentinvention are not synthetic, but are naturally-occurring entities thathave been isolated from their natural environments.

Small Molecule Targeting Moieties. In some embodiments, a targetingmoiety in accordance with the present invention may be a small molecule.In certain embodiments, small molecules are less than about 2000 g/molin size. In some embodiments, small molecules are less than about 1500g/mol or less than about 1000 g/mol. In some embodiments, smallmolecules are less than about 800 g/mol or less than about 500 g/mol.

In certain embodiments, a small molecule is oligomeric. In certainembodiments, a small molecule is non-oligomeric. In certain embodiments,a small molecule is a natural product or a natural product-like compoundhaving a partial structure (e.g., a substructure) based on the fullstructure of a natural product. In certain embodiments, a small moleculeis a synthetic product. In some embodiments, a small molecule may befrom a chemical library. In some embodiments, a small molecule may befrom a pharmaceutical company historical library. In certainembodiments, a small molecule is a drug approved by the U.S. Food andDrug Administration as provided in the U.S. Code of Federal Regulations(C.F.R.).

One of ordinary skill in the art will appreciate that any small moleculethat specifically binds to a desired target, as described herein, can beused in accordance with the present invention.

Protein Targeting Moieties. In some embodiments, a targeting moiety inaccordance with the present invention may be a protein or peptide. Incertain embodiments, peptides range from about 5 to about 100, fromabout 5 to about 50, from about 10 to about 75, from about 15 to about50, or from about 20 to about 25 amino acids in size. In someembodiments, a peptide sequence can be based on the sequence of aprotein. In some embodiments, a peptide sequence can be a randomarrangement of amino acids.

The terms “polypeptide” and “peptide” are used interchangeably herein,with “peptide” typically referring to a polypeptide having a length ofless than about 100 amino acids. Polypeptides may contain L-amino acids,D-amino acids, or both and may contain any of a variety of amino acidmodifications or analogs known in the art. Useful modifications include,e.g., terminal acetylation, amidation, lipidation, phosphorylation,glycosylation, acylation, farnesylation, sulfation, etc.

Exemplary proteins that may be used as targeting moieties in accordancewith the present invention include, but are not limited to, antibodies,receptors, cytokines, peptide hormones, glycoproteins, glycopeptides,proteoglycans, proteins derived from combinatorial libraries (e.g.,Avimers™, Affibodies®, etc.), and characteristic portions thereof.Synthetic binding proteins such as Nanobodies™, AdNectins™, etc., can beused. In some embodiments, protein targeting moieties can be peptides.

One of ordinary skill in the art will appreciate that any protein and/orpeptide that specifically binds to a desired target, as describedherein, can be used in accordance with the present invention.

In some embodiments, a targeting moiety may be an antibody and/orcharacteristic portion thereof. The term “antibody” refers to anyimmunoglobulin, whether natural or wholly or partially syntheticallyproduced and to derivatives thereof and characteristic portions thereof.An antibody may be monoclonal or polyclonal. An antibody may be a memberof any immunoglobulin class, including any of the human classes: IgG,IgM, IgA, IgD, and IgE.

As used herein, an antibody fragment (i.e. characteristic portion of anantibody) refers to any derivative of an antibody which is less thanfull-length. In some embodiments, an antibody fragment retains at leasta significant portion of the full-length antibody's specific bindingability. Examples of such antibody fragments include, but are notlimited to, Fab, Fab′, F(ab′)2, scFv, Fv, dsFv diabody, and Fdfragments. Antibody fragments also include, but are not limited, to Fcfragments.

An antibody fragment may be produced by any means. For example, anantibody fragment may be enzymatically or chemically produced byfragmentation of an intact antibody and/or it may be recombinantlyproduced from a gene encoding the partial antibody sequence.Alternatively or additionally, an antibody fragment may be wholly orpartially synthetically produced. An antibody fragment may optionallycomprise a single chain antibody fragment. Alternatively oradditionally, an antibody fragment may comprise multiple chains whichare linked together, for example, by disulfide linkages. An antibodyfragment may optionally comprise a multimolecular complex. A functionalantibody fragment will typically comprise at least about 50 amino acidsand more typically will comprise at least about 200 amino acids.

In some embodiments, antibodies may include chimeric (e.g. “humanized”)and single chain (recombinant) antibodies. In some embodiments,antibodies may have reduced effector functions and/or bispecificmolecules. In some embodiments, antibodies may include fragmentsproduced by a Fab expression library.

Single-chain Fvs (scFvs) are recombinant antibody fragments consistingof only the variable light chain (VL) and variable heavy chain (VH)covalently connected to one another by a polypeptide linker. Either VLor VH may comprise the NH2-terminal domain. The polypeptide linker maybe of variable length and composition so long as the two variabledomains are bridged without significant steric interference. Typically,linkers primarily comprise stretches of glycine and serine residues withsome glutamic acid or lysine residues interspersed for solubility.

Diabodies are dimeric scFvs. Diabodies typically have shorter peptidelinkers than most scFvs, and they often show a preference forassociating as dimers.

An Fv fragment is an antibody fragment which consists of one VH and oneVL domain held together by noncovalent interactions. The term “dsFv” asused herein refers to an Fv with an engineered intermolecular disulfidebond to stabilize the VH-VL pair.

An F(ab′)2 fragment is an antibody fragment essentially equivalent tothat obtained from immunoglobulins by digestion with an enzyme pepsin atpH 4.0-4.5. The fragment may be recombinantly produced.

A Fab′ fragment is an antibody fragment essentially equivalent to thatobtained by reduction of the disulfide bridge or bridges joining the twoheavy chain pieces in the F(ab′)2 fragment. The Fab′ fragment may berecombinantly produced.

A Fab fragment is an antibody fragment essentially equivalent to thatobtained by digestion of immunoglobulins with an enzyme (e.g., papain).The Fab fragment may be recombinantly produced. The heavy chain segmentof the Fab fragment is the Fd piece.

Carbohydrate Targeting Moieties. In some embodiments, a targeting moietyin accordance with the present invention may comprise a carbohydrate. Insome embodiments, a carbohydrate may be a polysaccharide comprisingsimple sugars (or their derivatives) connected by glycosidic bonds, asknown in the art. Such sugars may include, but are not limited to,glucose, fructose, galactose, ribose, lactose, sucrose, maltose,trehalose, cellbiose, mannose, xylose, arabinose, glucoronic acid,galactoronic acid, mannuronic acid, glucosamine, galatosamine, andneuramic acid. In some embodiments, a carbohydrate may be one or more ofpullulan, cellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, hydroxycellulose, methylcellulose, dextran,cyclodextran, glycogen, starch, hydroxyethylstarch, carageenan, glycon,amylose, chitosan, N,O-carboxylmethylchitosan, algin and alginic acid,starch, chitin, heparin, konjac, glucommannan, pustulan, heparin,hyaluronic acid, curdlan, and xanthan.

In some embodiments, the carbohydrate may be aminated, carboxylated,and/or sulfated. In some embodiments, hydrophilic polysaccharides can bemodified to become hydrophobic by introducing a large number ofside-chain hydrophobic groups. In some embodiments, a hydrophobiccarbohydrate may include cellulose acetate, pullulan acetate, konjacacetate, amylose acetate, and dextran acetate.

One of ordinary skill in the art will appreciate that any carbohydratethat specifically binds to a desired target, as described herein, can beused in accordance with the present invention.

Lipid Targeting Moieties. In some embodiments, a targeting moiety inaccordance with the present invention may comprise one or more fattyacid groups or salts thereof. In some embodiments, a fatty acid groupmay comprise digestible, long chain (e.g., C₈-C₅₀), substituted orunsubstituted hydrocarbons. In some embodiments, a fatty acid group maybe a C₁₀-C₂₀ fatty acid or salt thereof. In some embodiments, a fattyacid group may be a C₁₅-C₂₀ fatty acid or salt thereof. In someembodiments, a fatty acid group may be a C₁₅-C₂₅ fatty acid or saltthereof. In some embodiments, a fatty acid group may be unsaturated.

In some embodiments, a fatty acid group may be monounsaturated. In someembodiments, a fatty acid group may be polyunsaturated. In someembodiments, a double bond of an unsaturated fatty acid group may be inthe cis conformation. In some embodiments, a double bond of anunsaturated fatty acid may be in the trans conformation.

In some embodiments, a fatty acid group may be one or more of butyric,caproic, caprylic, capric, lauric, myristic, palmitic, stearic,arachidic, behenic, or lignoceric acid. In some embodiments, a fattyacid group may be one or more of palmitoleic, oleic, vaccenic, linoleic,alpha-linoleic, gamma-linoleic, arachidonic, gadoleic, arachidonic,eicosapentaenoic, docosahexaenoic, or erucic acid.

One of ordinary skill in the art will appreciate that any fatty acidgroup that specifically binds to a desired target, as described herein,can be used in accordance with the present invention.

Novel Targeting Moieties

Any novel targeting moiety can be utilized in the nanocarriers inaccordance with the present invention. Any method known in the art canbe used to design, identify, and/or isolate novel targeting moieties.For example, standard techniques utilizing libraries of molecules and invitro binding assays can be utilized to identify novel targetingmoieties.

Nucleic acid targeting moieties (e.g. aptamers, Spiegelmers®) may bedesigned and/or identified using any available method. In someembodiments, nucleic acid targeting moieties are designed and/oridentified by identifying nucleic acid targeting moieties from acandidate mixture of nucleic acids. Systemic Evolution of Ligands byExponential Enrichment (SELEX), or a variation thereof, is a commonlyused method of identifying nucleic acid targeting moieties that bind toa target from a candidate mixture of nucleic acids (see, e.g., U.S. Pat.Nos. 6,482,594; 6,458,543; 6,458,539; 6,376,190; 6,344,318; 6,242,246;6,184,364; 6,001,577; 5,958,691; 5,874,218; 5,853,984; 5,843,732;5,843,653; 5,817,785; 5,789,163; 5,763,177; 5,696,249; 5,660,985;5,595,877; 5,567,588; and 5,270,163; each of which is incorporatedherein by reference). Alternatively or additionally, Polyplex In VivoCombinatorial Optimization (PICO) is a method that can be used toidentify nucleic acid targeting moieties (e.g. aptamers) that bind to atarget from a candidate mixture of nucleic acids in vivo and/or in vitroand is described in co-pending PCT Application US06/47975, entitled“System for Screening Particles,” filed Dec. 15, 2006, which isincorporated herein by reference.

Immunostimulatory Agents

In some embodiments, nanocarriers may transport one or moreimmunostimulatory agents which can help stimulate immune responses. Insome embodiments, immunostimulatory agents boost immune responses byactivating APCs to enhance their immunostimulatory capacity. In someembodiments, immunostimulatory agents boost immune responses byamplifying lymphocyte responses to specific antigens. In someembodiments, immunostimulatory agents boost immune responses by inducingthe local release of mediators, such as cytokines from a variety of celltypes. In some embodiments, the immunostimulatory agents suppress orredirect an immune response. In some embodiments, the immunostimulatoryagents induce regulatory T cells.

In some embodiments, all of the immunostimulatory agents of a vaccinenanocarrier are identical to one another. In some embodiments, a vaccinenanocarrier comprises a number of different types of immunostimulatoryagents. In some embodiments, a vaccine nanocarrier comprises multipleindividual immunostimulatory agents, all of which are identical to oneanother. In some embodiments, a vaccine nanocarrier comprises exactlyone type of immunostimulatory agent. In some embodiments, a vaccinenanocarrier comprises exactly two distinct types of immunostimulatoryagents. In some embodiments, a vaccine nanocarrier comprises greaterthan two distinct types of immunostimulatory agents.

In some embodiments, a vaccine nanocarrier comprises a single type ofimmunostimulatory agent that stimulates both B cells and T cells. Insome embodiments, a vaccine nanocarrier comprises two types ofimmunostimulatory agents, wherein first type of immunostimulatory agentstimulates B cells, and the second type of immunostimulatory agentstimulates T cells. In some embodiments, a vaccine nanocarrier comprisesgreater than two types of immunostimulatory agents, wherein one or moretypes of immunostimulatory agents stimulate B cells, and one or moretypes of immunostimulatory agents stimulate T cells.

In some embodiments, a vaccine nanocarrier comprises at least one typeof immunostimulatory agent that is associated with the exterior surfaceof the vaccine nanocarrier. In some embodiments, the association iscovalent. In some embodiments, the covalent association is mediated byone or more linkers. In some embodiments, the association isnon-covalent. In some embodiments, the non-covalent association ismediated by charge interactions, affinity interactions, metalcoordination, physical adsorption, host-guest interactions, hydrophobicinteractions, TT stacking interactions, hydrogen bonding interactions,van der Waals interactions, magnetic interactions, electrostaticinteractions, dipole-dipole interactions, and/or combinations thereof.Association of immunostimulatory agents with vaccine nanocarriers isdescribed in further detail below, in the section entitled “Productionof Vaccine Nanocarriers.”

In some embodiments, a vaccine nanocarrier comprises a lipid membrane(e.g., lipid bilayer, lipid monolayer, etc.), wherein at least one typeof immunostimulatory agent is associated with the lipid membrane. Insome embodiments, at least one type of immunostimulatory agent isembedded within the lipid membrane. In some embodiments, at least onetype of immunostimulatory agent is embedded within the lumen of a lipidbilayer. In some embodiments, a vaccine nanocarrier comprises at leastone type of immunostimulatory agent that is associated with the interiorsurface of the lipid membrane. In some embodiments, at least one type ofimmunostimulatory agent is encapsulated with the lipid membrane of avaccine nanocarrier. In some embodiments, at least one type ofimmunostimulatory agent may be located at multiple locations of avaccine nanocarrier. For example, a first type of immunostimulatoryagent may be embedded within a lipid membrane, and a second type ofimmunostimulatory agent may be encapsulated within the lipid membrane ofa vaccine nanocarrier. To give another example, a first type ofimmunostimulatory agent may be associated with the exterior surface of alipid membrane, and a second type of immunostimulatory agent may beassociated with the interior surface of the lipid membrane of a vaccinenanocarrier. In some embodiments, a first type of immunostimulatoryagent may be embedded within the lumen of a lipid bilayer of a vaccinenanocarrier, and the lipid bilayer may encapsulate a polymeric matrixthroughout which a second type of immunostimulatory agent isdistributed. In some embodiments, a first type of immunostimulatoryagent and a second type of immunostimulatory agent may be in the samelocale of a vaccine nanocarrier (e.g., they may both be associated withthe exterior surface of a vaccine nanocarrier; they may both beencapsulated within the vaccine nanocarrier; etc.). One of ordinaryskill in the art will recognize that the preceding examples are onlyrepresentative of the many different ways in which multipleimmunostimulatory agents may be associated with different locales ofvaccine nanocarriers. Multiple immunostimulatory agents may be locatedat any combination of locales of vaccine nanocarriers.

In certain embodiments, immunostimulatory agents may be interleukins,interferon, cytokines, etc. In specific embodiments, animmunostimulatory agent may be a natural or synthetic agonist for aToll-like receptor (TLR). In specific embodiments, vaccine nanocarriersincorporate a ligand for toll-like receptor (TLR)-7, such as CpGs, whichinduce type I interferon production. In specific embodiments, animmunostimulatory agent may be an agonist for the DC surface moleculeCD40. In certain embodiments, to stimulate immunity rather thantolerance, a nanocarrier incorporates an immunostimulatory agent thatpromotes DC maturation (needed for priming of naive T cells) and theproduction of cytokines, such as type I interferons, which promoteantibody responses and anti-viral immunity. In some embodiments, animmunomodulatory agent may be a TLR-4 agonist, such as bacteriallipopolysaccharide (LPS), VSV-G, and/or HMGB-1. In some embodiments,immunomodulatory agents are cytokines, which are small proteins orbiological factors (in the range of 5 kD-20 kD) that are released bycells and have specific effects on cell-cell interaction, communicationand behavior of other cells. In some embodiments, immunostimulatoryagents may be proinflammatory stimuli released from necrotic cells(e.g., urate crystals). In some embodiments, immunostimulatory agentsmay be activated components of the complement cascade (e.g., CD21, CD35,etc.). In some embodiments, immunostimulatory agents may be activatedcomponents of immune complexes. The immunostimulatory agents includeTLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8, TLR-9, andTLR-10 agonists. The immunostimulatory agents also include complementreceptor agonists, such as a molecule that binds to CD21 or CD35. Insome embodiments, the complement receptor agonist induces endogenouscomplement opsonization of the nanocarrier. Immunostimulatory agentsalso include cytokine receptor agonists, such as a cytokine. In someembodiments, the cytokine receptor agonist is a small molecule,antibody, fusion protein, or aptamer.

In some embodiments, there are more than one type of immunostimulatoryagent. In some embodiments, the different immunostimulatory agents eachact on a different pathway. The immunostimulatory agents, therefore, canbe different Toll-like receptors, a Toll-like receptor and CD40, aToll-like receptor and a component of the inflammasome, etc.

In some embodiments, the immunostimulatory agent may be an adjuvant.Thus, in some embodiments, the present invention provides pharmaceuticalcompositions comprising vaccine nanocarriers formulated with one or moreadjuvants. The term “adjuvant”, as used herein, refers to an agent thatdoes not constitute a specific antigen, but boosts the immune responseto the administered antigen.

In some embodiments, vaccine nanocarriers are formulated with one ormore adjuvants such as gel-type adjuvants (e.g., aluminum hydroxide,aluminum phosphate, calcium phosphate, etc.), microbial adjuvants (e.g.,immunomodulatory DNA sequences that include CpG motifs; endotoxins suchas monophosphoryl lipid A; exotoxins such as cholera toxin, E. coli heatlabile toxin, and pertussis toxin; muramyl dipeptide, etc.);oil-emulsion and emulsifier-based adjuvants (e.g., Freund's Adjuvant,MF59 [Novartis], SAF, etc.); particulate adjuvants (e.g., liposomes,biodegradable microspheres, saponins, etc.); synthetic adjuvants (e.g.,nonionic block copolymers, muramyl peptide analogues, polyphosphazene,synthetic polynucleotides, etc.), surfactant based adjuvants, and/orcombinations thereof. Other exemplary adjuvants include some polymers(e.g., polyphosphazenes, described in U.S. Pat. No. 5,500,161, which isincorporated herein by reference), QS21, squalene, tetrachlorodecaoxide,etc.

Assays for T Cell Activation

In some embodiments, various assays can be utilized in order todetermine whether an immune response has been stimulated in a T cell orgroup of T cells (i.e., whether a T cell or group of T cells has become“activated”). In some embodiments, stimulation of an immune response inT cells can be determined by measuring antigen-induced production ofcytokines by T cells. In some embodiments, stimulation of an immuneresponse in T cells can be determined by measuring antigen-inducedproduction of IFNγ, IL-4, IL-2, IL-10, IL-17 and/or TNFα by T cells. Insome embodiments, antigen-produced production of cytokines by T cellscan be measured by intracellular cytokine staining followed by flowcytometry. In some embodiments, antigen-induced production of cytokinesby T cells can be measured by surface capture staining followed by flowcytometry. In some embodiments, antigen-induced production of cytokinesby T cells can be measured by determining cytokine concentration insupernatants of activated T cell cultures. In some embodiments, this canbe measured by ELISA.

In some embodiments, antigen-produced production of cytokines by T cellscan be measured by ELISPOT assay. In general, ELISPOT assays employ atechnique very similar to the sandwich enzyme-linked immunosorbent assay(ELISA) technique. An antibody (e.g. monoclonal antibody, polyclonalantibody, etc.) is coated aseptically onto a PVDF (polyvinylidenefluoride)-backed microplate. Antibodies are chosen for their specificityfor the cytokine in question. The plate is blocked (e.g. with a serumprotein that is non-reactive with any of the antibodies in the assay).Cells of interest are plated out at varying densities, along withantigen or mitogen, and then placed in a humidified 37° C. CO₂ incubatorfor a specified period of time. Cytokine secreted by activated cells iscaptured locally by the coated antibody on the high surface area PVDFmembrane. After washing the wells to remove cells, debris, and mediacomponents, a secondary antibody (e.g., a biotinylated polyclonalantibody) specific for the cytokine is added to the wells. This antibodyis reactive with a distinct epitope of the target cytokine and thus isemployed to detect the captured cytokine. Following a wash to remove anyunbound biotinylated antibody, the detected cytokine is then visualizedusing an avidin-HRP, and a precipitating substrate (e.g., AEC,BCIP/NBT). The colored end product (a spot, usually a blackish blue)typically represents an individual cytokine-producing cell. Spots can becounted manually (e.g., with a dissecting microscope) or using anautomated reader to capture the microwell images and to analyze spotnumber and size. In some embodiments, each spot correlates to a singlecytokine-producing cell.

In some embodiments, an immune response in T cells is said to bestimulated if between about 1% and about 100% of antigen-specific Tcells produce cytokines. In some embodiments, an immune response in Tcells is said to be stimulated if at least about 1%, at least about 5%,at least about 10%, at least about 25%, at least about 50%, at leastabout 75%, at least about 90%, at least about 95%, at least about 99%,or about 100% of antigen-specific T cells produce cytokines.

In some embodiments, an immune response in T cells is said to bestimulated if immunized subjects comprise at least about 10-fold, atleast about 50-fold, at least about 100-fold, at least about 500-fold,at least about 1000-fold, at least about 5000-fold, at least about10,000-fold, at least about 50,000-fold, at least about 100,000-fold, orgreater than at least about 100,000-fold more cytokine-producing cellsthan do naive controls.

In some embodiments, stimulation of an immune response in T cells can bedetermined by measuring antigen-induced proliferation of T cells. Insome embodiments, antigen-induced proliferation may be measured asuptake of H³-thymidine in dividing T cells (sometimes referred to as“lymphocyte transformation test, or “LTT”). In some embodiments,antigen-induced proliferation is said to have occurred if H³-thymidineuptake (given as number of counts from a γ counter) is at least about5-fold, at least about 10-fold, at least about 20-fold, at least about50-fold, at least about 100-fold, at least about 500-fold, at leastabout 1000-fold, at least about 5000-fold, at least about 10,000-fold,or greater than at least about 10,000-fold higher than a naïve control.

In some embodiments, antigen-induced proliferation may be measured byflow cytometry. In some embodiments, antigen-induced proliferation maybe measured by a carboxyfluorescein succinimidyl ester (CFSE) dilutionassay. CFSE is a non-toxic, fluorescent, membrane-permeating dye thatbinds the amino groups of cytoplasmic proteins with itssuccinimidyl-reactive group (e.g. T cell proteins). When cells divide,CFSE-labeled proteins are equally distributed between the daughtercells, thus halving cell fluorescence with each division. Consequently,antigen-specific T cells lose their fluorescence after culture in thepresence of the respective antigen (CFSE^(low)) and are distinguishablefrom other cells in culture (CFSE^(high)). In some embodiments,antigen-induced proliferation is said to have occurred if CFSE dilution(given as the percentage of CFSE^(low) cells out of all CFSE⁺ cells) isat least about 5%, at least about 10%, at least about 25%, at leastabout 50%, at least about 75%, at least about 90%, at least about 95%,or at least about 100%.

In some embodiments, an immune response in T cells is said to bestimulated if cellular markers of T cell activation are expressed atdifferent levels (e.g. higher or lower levels) relative to unstimulatedcells. In some embodiments, CD11a CD27, CD25, CD40L, CD44, CD45RO,and/or CD69 are more highly expressed in activated T cells than inunstimulated T cells. In some embodiments, L-selectin (CD62L), CD45RA,and/or CCR7 are less highly expressed in activated T cells than inunstimulated T cells.

In some embodiments, an immune response in T cells is measured byassaying cytotoxicity by effector CD8⁺ T cells against antigen-pulsedtarget cells. For example, a ⁵¹chromium (⁵¹Cr) release assay can beperformed. In this assay, effector CD8⁺ T cells bind infected cellspresenting virus peptide on class I MHC and signal the infected cells toundergo apoptosis. If the cells are labeled with ⁵¹Cr before theeffector CD8⁺T cells are added, the amount of ⁵¹Cr released into thesupernatant is proportional to the number of targets killed.

One of ordinary skill in the art will recognize that the assaysdescribed above are only exemplary methods which could be utilized inorder to determine whether T cell activation has occurred. Any assayknown to one of skill in the art which can be used to determine whetherT cell activation has occurred falls within the scope of this invention.The assays described herein as well as additional assays that could beused to determine whether T cell activation has occurred are describedin Current Protocols in Immunology (John Wiley & Sons, Hoboken, N.Y.,2007; incorporated herein by reference).

B Cells

The present invention provides vaccine nanocarriers for delivery of, forexample, immunomodulatory agents to the cells of the immune system. Insome embodiments, vaccine nanocarriers comprise at least oneimmunomodulatory agent which can be presented to B cells (i.e., B cellantigens).

Immunomodulatory Agents

B cells and T cells recognize antigen by different mechanisms. Asdescribed above, T cells recognize antigen in a processed form (e.g., asa peptide fragment presented by an APC's MHC molecule to the T cellreceptor). B cells recognize antigens in their native form. B cellsrecognize free (e.g., soluble) antigen in blood or lymph using B cellreceptors (BCRs) and/or membrane bound-immunoglobulins.

The immunomodulatory agent can be a B cell antigen. B cell antigensinclude, but are not limited to proteins, peptides, small molecules, andcarbohydrates. In some embodiments, the B cell antigen is a non-proteinantigen (i.e., not a protein or peptide antigen). In some embodiments,the B cell antigen is a carbohydrate associated with an infectiousagent. In some embodiments, the B cell antigen is a glycoprotein orglycopeptide associated with an infectious agent. The infectious agentcan be a bacterium, virus, fungus, protozoan, or parasite. In someembodiments, the B cell antigen is a poorly immunogenic antigen. In someembodiments, the B cell antigen is an abused substance or a portionthereof. In some embodiments, the B cell antigen is an addictivesubstance or a portion thereof.

Addictive substances include, but are not limited to, drugs known tocause addiction including stimulants, sedatives and opiates,amphetamine, cocaine, heroine, caffeine, alcohol, barbiturates,benzodiazepines, methaqualone, morphine, phentanyl, solvents, anabolicsteroids, cannabis, nicotine, narcotics, cough suppressants,tranquilizers, sedatives and all metabolites, analogues, and derivativesthereof. Further examples of addictive substances include those providedelsewhere herein.

In some embodiments, the addictive substance comprises one or morechiral carbon centers, and may, accordingly, be present in anenantiomerically pure form or a mixture of isomers. In some embodiments,where the addictive qualities of the addictive substance are dependenton the stereochemistry of the ciral carbon(s), the addictive substanceused in the compositions described herein is present as the isomer thatis most addictive, or as the isomer that is most commonly available, oras the isomer that is most commonly responsible for addiction amongusers.

For example, the addictive substance may be nicotine. In some preferredembodiments, for example, the addictive substance may be(S)-(−)-nicotine or (R)-(−)-nicotine. In preferred embodiments, theaddictive substance is (S)-(−)-nicotine.

Drug addiction is considered a pathological state, involving theprogression of acute drug use to the development of drug-seekingbehavior, the vulnerability to relapse, and the decreased, slowedability to respond to naturally rewarding stimuli. The Diagnostic andStatistical Manual of Mental Disorders, Fourth Edition (DSM-IV) hascategorized three stages of addiction: preoccupation/anticipation,bingelintoxication, and withdrawal/negative affect. These stages arecharacterized, respectively, everywhere by constant cravings andpreoccupation with obtaining the substance; using more of the substancethan necessary to experience the intoxicating effects; and experiencingtolerance, withdrawal symptoms, and decreased motivation for normal lifeactivities. By the American Society of Addiction Medicine definition,drug addiction differs from drug dependence and drug tolerance. The termdrug addiction is also used as a category which may include the samepersons who can be given the diagnosis of substance dependence orsubstance abuse.

In some embodiments, the B cell antigen is a toxin, such as a toxin froma chemical weapon. In some embodiments, the toxin from a chemical weaponis botulinum toxin or phosphene. Toxins from a chemical weapon include,but are not limited to, O-Alkyl (<C10, incl. cycloalkyl) alkyl (Me, Et,n-Pr or i-Pr)-phosphonofluoridates (e.g. Sarin: O-Isopropylmethylphosphonofluoridate, Soman: O-Pinacolylmethylphosphonofluoridate), O-Alkyl (<C10, incl. cycloalkyl) N,N-dialkyl(Me, Et, n-Pr or i-Pr) phosphoramidocyanidates (e.g. Tabun: O-EthylN,N-dimethylphosphoramidocyanidate), O-Alkyl (H or <C10, incl.cycloalkyl) S-2-dialkyl (Me, Et, n-Pr or i-Pr)-aminoethyl alkyl (Me, Et,n-Pr or i-Pr) phosphonothiolates and corresponding alkylated orprotonated salts (e.g. VX: O-Ethyl S-2-diisopropylaminoethylmethylphosphonothiolate), Sulfur mustards:2-Chloroethylchloromethylsulfide, Mustard gas:Bis(2-chloroethyl)sulfide, Bis(2-chloroethylthio)methane, Sesquimustard:1,2-Bis(2-chloroethylthio)ethane, 1,3-Bis(2-chloroethylthio)-n-propane,1,4-Bis(2-chloroethylthio)-n-butane,1,5-Bis(2-chloroethylthio)-n-pentane, Bis(2-chloroethylthiomethyl)ether,O-Mustard: Bis(2-chloroethylthioethyl)ether, Lewisites: Lewisite 1:2-Chlorovinyldichloroarsine, Lewisite 2: Bis(2-chlorovinyl)chloroarsine,Lewisite 3: Tris(2-chlorovinyl)arsine, Nitrogen mustards: HN1:Bis(2-chloroethyl)ethylamine, HN2: Bis(2-chloroethyl)methylamine, HN3:Tris(2-chloroethyl)amine, Saxitoxin, Ricin, Amiton: O,O-DiethylS-(2-(diethylamino)ethyl)phosphorothiolate and corresponding alkylatedor protonated salts, PFIB:1,1,3,3,3-Pentafluoro-2-(trifluoromethyl)-1-propene, 3-Quinuclidinylbenzilate (BZ), Phosgene: Carbonyl dichloride, Cyanogen chloride,Hydrogen cyanide and Chloropicrin: Trichloronitromethane.

The B cell antigen may also be a hazardous environmental agent.Hazardous environmental agents include, but are not limited to, arsenic,lead, mercury, vinyl chloride, polychlorinated biphenyls, benzene,polycyclic aromatic hydrocarbons, cadmium, benzo(a)pyrene,benzo(b)fluoranthene, chloroform, DDT, P,P′-, aroclor 1254, aroclor1260, dibenzo(a,h)anthracene, trichloroethylene, dieldrin, chromiumhexavalent, and DDE, P,P′. Examples of such agents include thoseprovided elsewhere herein.

In some embodiments, the B cell antigen is a self antigen. In otherembodiments, the B cell antigen is an alloantigen, a contact sensitizer,a degenerative disease antigen, a hapten, an infectious disease antigen,a cancer antigen, an atopic disease antigen, an autoimmune diseaseantigen, an addictive substance, a xenoantigen, or a metabolic diseaseenzyme or enzymatic product thereof. Examples of such antigens includethose provided elsewhere herein.

As described above, the present invention provides vaccine nanocarrierscomprising, for example, one or more immunomodulatory agents. In someembodiments, inventive nanocarriers comprising one or moreimmunomodulatory agents are used as vaccines. In some embodiments,antigen presentation to B cells can be optimized by presentingstructurally intact immunomodulatory agents on the surface ofnanocarriers. In some embodiments, structurally intact immunomodulatoryagents are presented on the surface of vaccine nanocarriers at high copynumber and/or density.

In some embodiments, an immunomodulatory agent may comprise isolatedand/or recombinant proteins or peptides, inactivated organisms andviruses, dead organisms and virus, genetically altered organisms orviruses, and cell extracts. In some embodiments, an immunomodulatoryagent may comprise nucleic acids, carbohydrates, lipids, and/or smallmolecules. In some embodiments, an immunomodulatory agent is one thatelicits an immune response. In some embodiments, an immunomodulatoryagent is an antigen. In some embodiments, an immunomodulatory agent isused for vaccines. Further description of immunomodulatory agents can befound in the section above entitled “B Cells.”

As discussed above, a vaccine nanocarrier may comprise a single type ofimmunomodulatory agent that stimulates both B cells and T cells. In someembodiments, a vaccine nanocarrier comprises two types ofimmunomodulatory agents, wherein first type of immunomodulatory agentstimulates B cells, and the second type of immunomodulatory agentstimulates T cells. In some embodiments, a vaccine nanocarrier comprisesgreater than two types of immunomodulatory agents, wherein one or moretypes of immunomodulatory agents stimulate B cells, and one or moretypes of immunomodulatory agents stimulate T cells.

Targeting Moieties

As discussed above, inventive nanocarriers comprise one or moretargeting moieties. For a discussion of general and specific propertiesof targeting moieties in accordance with the present invention, see thesubheading entitled “Targeting Moieties” in the section above entitled“T Cells.” In some embodiments, targeting moieties target particularcell types. In certain embodiments, a target is a B cell marker. In someembodiments, a B cell target is an antigen that is expressed in B cellsbut not in non-B cells. In some embodiments, a B cell target is anantigen that is more prevalent in B cells than in non-B cells.

In certain embodiments, a target is a SCS-Mph marker. In someembodiments, an SCS-Mph target is an antigen that is expressed inSCS-Mph but not in non-SCS-Mph. In some embodiments, an SCS-Mph targetis an antigen that is more prevalent in SCS-Mph than in non-SCS-Mph.Exemplary SCS-Mph markers are listed below in the section entitled“Subcapsular Sinus Macrophage Cells” and include those providedelsewhere herein. In some embodiments, when the target is a SCS-Mph, thetargeting moiety on the vaccine nanocarriers is nicotine, or aderivative or analogue of nicotine (such as a fragment of nicotine).Examples of nanocarriers having nicotine targeting moieties includeinstances where nicotine is associated (such as through a covalent bond)with one of the components of the nanocarriers. One example is anicotine-polymer conjugate, wherein nicotine is covalently bound to apolymer molecule that is part of the nanocarrier.

In certain embodiments, a target is a FDC marker. In some embodiments,an FDC target is an antigen that is expressed in FDCs but not innon-FDCs. In some embodiments, an FDC target is an antigen that is moreprevalent in FDCs than in non-FDCs. Exemplary FDC markers are listedbelow in the section entitled “Follicular Dendritic Cells” and includethose provided elsewhere herein.

In some embodiments, a target is preferentially expressed in particularcell types. For example, expression of an SCS-Mph, FDC, and/or B celltarget in SCS-Mph, FDCs, and/or B cells is at least 2-fold, at least3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least20-fold, at least 50-fold, at least 100-fold, at least 500-fold, or atleast 1000-fold overexpressed in SCS-Mph, FDCs, and/or B cells relativeto a reference population. In some embodiments, a reference populationmay comprise non-SCS-Mph, FDCs, and/or B cells.

In some embodiments, expression of an SCS-Mph, FDC, and/or B cell targetin activated SCS-Mph, FDCs, and/or B cells is at least 2-fold, at least3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least20-fold, at least 50-fold, at least 100-fold, at least 500-fold, or atleast 1000-fold overexpressed in activated SCS-Mph, FDCs, and/or B cellsrelative to a reference population. In some embodiments, a referencepopulation may comprise non-activated SCS-Mph, FDCs, and/or B cells.

Subcapsular Sinus Macrophage Cells

The present invention encompasses the recognition that targeting ofantigens to subcapsular sinus macrophages (SCS-Mph) is involved inefficient early presentation of lymph-borne pathogens, such as viruses,to follicular B cells (FIG. 2). As described in Example 1, followingsubcutaneous injection of vesicular stomatitis virus (VSV) or adenovirus(AdV) into the footpad of mice, viral particles were efficiently andselectively retained by CD169⁺SCS-Mph in the draining popliteal lymphnodes. VSV-specific B cell receptor (BCR) transgenic B cells in theselymph nodes were rapidly activated and generated extremely high antibodytiters upon this viral challenge. Depletion of SCS-Mph by injection ofliposomes laden with clodronate (which is toxic for Mph) abolished earlyB cell activation, indicating that SCS-Mph are essential for thepresentation of lymph-borne particulate antigens to B cells.

B cells are more potently activated by polyvalent antigens that arepresented to them on a fixed surface, rather than in solution. While notwishing to be bound by any one theory, the present invention suggests areason why many enveloped viruses (such as VSV) elicit potentneutralizing antibody responses to their envelope glycoprotein: theantigenic protein is presented at a very high density on the surface ofthe viral particles, and the viral particles are presented to B cells ina relatively immobile manner, i.e., bound to the plasma membrane ofSCS-Mph. The present invention encompasses the recognition that vaccinecarriers that mimic viral particles by targeting SCS-Mph uponsubcutaneous injection and presenting polyvalent conformationally intactantigens on their surface can stimulate a potent B cell response.

In some embodiments, SCS-Mph targeting is accomplished by moieties thatbind CD169 (i.e., sialoadhesin), CD11b (i.e., CD11b/CD18, Mac-1, CR3 orαMβ2 integrin), and/or the mannose receptor (i.e., a multi-valentlectin), proteins which are all prominently expressed on SCS-Mph.Examples of such moieties include those provided elsewhere herein.

In some embodiments, SCS-Mph targeting can be accomplished by anytargeting moiety that specifically binds to any entity (e.g., protein,lipid, carbohydrate, small molecule, etc.) that is prominently expressedand/or present on macrophages (i.e., SCS-Mph markers). Exemplary SCS-Mphmarkers include, but are not limited to, CD4 (L3T4, W3/25, T4); CD9(p24, DRAP-1, MRP-1); CD11a (LFA-1α, αL Integrin chain); CD11b (αMIntegrin chain, CR3, Mo1, C3niR, Mac-1); CD11c (αX Integrin, p150, 95,AXb2); CDw12 (p90-120); CD13 (APN, gp150, EC 3.4.11.2); CD14 (LPS-R);CD15 (X-Hapten, Lewis, X, SSEA-1,3-FAL); CD15s (Sialyl Lewis X); CD15u(3′ sulpho Lewis X); CD15su (6 sulpho-sialyl Lewis X); CD16a (FCRIIIA);CD16b (FcgRIIIb); CDw17 (Lactosylceramide, LacCer); CD18 (Integrin β2,CD11a,b,c β-subunit); CD26 (DPP IV ectoeneyme, ADA binding protein);CD29 (Platelet GPIIa, β-1 integrin, GP); CD31 (PECAM-1, Endocam); CD32(FCγRII); CD33 (gp67); CD35 (CR1, C3b/C4b receptor); CD36 (GpIIIb, GPIV,PASIV); CD37 (gp52-40); CD38 (ADP-ribosyl cyclase, T10); CD39(ATPdehydrogenase, NTPdehydrogenase-1); CD40 (Bp50); CD43 (Sialophorin,Leukosialin); CD44 (EMCRII, H-CAM, Pgp-1); CD45 (LCA, T200, B220, Ly5);CD45RA; CD45RB; CD45RC; CD45RO (UCHL-1); CD46 (MCP); CD47 (gp42, IAP,OA3, Neurophillin); CD47R (MEM-133); CD48 (Blast-1, Hulym3, BCM-1,OX-45); CD49a (VLA-1α, α1 Integrin); CD49b (VLA-2α, gp1a, α2 Integrin);CD49c (VLA-3α, α3 Integrin); CD49e (VLA-5α, α5 Integrin); CD49f (VLA-6α,α6 Integrin, gplc); CD50 (ICAM-3); CD51 (Integrin α, VNR-α,Vitronectin-Rα); CD52 (CAMPATH-1, HE5); CD53 (OX-44); CD54 (ICAM-1);CD55 (DAF); CD58 (LFA-3); CD59 (1F5Ag, H19, Protectin, MACIF, MIRL,P-18); CD60a (GD3); CD60b (9-O-acetyl GD3); CD61 (GP IIIa, β3 Integrin);CD62L (L-selectin, LAM-1, LECAM-1, MEL-14, Leu8, TQ1); CD63 (LIMP, MLA1,gp55, NGA, LAMP-3, ME491); CD64 (FcγRI); CD65 (Ceramide, VIM-2); CD65s(Sialylated-CD65, VIM2); CD72 (Ly-19.2, Ly-32.2, Lyb-2); CD74 (Ii,invariant chain); CD75 (sialo-masked Lactosamine); CD75S (α2,6sialylated Lactosamine); CD80 (B7, B7-1, BB1); CD81 (TAPA-1); CD82 (4F9,C33, IA4, KAI1, R2); CD84 (p75, GR6); CD85a (ILT5, LIR2, HL9); CD85d(ILT4, LIR2, MIR10); CD85j (ILT2, LIR1, MIR7); CD85k (ILT3, LIR5, HM18);CD86 (B7-2/B70); CD87 (uPAR); CD88 (C5aR); CD89 (IgA Fc receptor, FcαR);CD91 (α2M-R, LRP); CDw92 (p70); CDw93 (GR11); CD95 (APO-1, FAS,TNFRSF6); CD97 (BL-KDD/F12); CD98 (4F2, FRP-1, RL-388); CD99 (MIC2, E2);CD99R (CD99 Mab restricted); CD100 (SEMA4D); CD101 (IGSF2, P126, V7);CD102 (ICAM-2); CD111 (PVRL1, HveC, PRR1, Nectin 1, HIgR); CD112 (HveB,PRR2, PVRL2, Nectin2); CD114 (CSF3R, G-CSRF, HG-CSFR); CD115 (c-fms,CSF-1R, M-CSFR); CD116 (GM-CSFRα); CDw119 (IFNγR, IFNγRA); CD120a(TNFRI, p55); CD120b (TNFRII, p75, TNFR p80); CD121b (Type 2 IL-1R);CD122 (IL2Rβ); CD123 (IL-3Rα); CD124 (IL-4Rα); CD127 (p90, IL-7R,IL-7Rα); CD128a (IL-8Ra, CXCR1, (Tentatively renamed as CD181)); CD128b(IL-8Rb, CSCR2, (Tentatively renamed as CD182)); CD130 (gp130); CD131(Common β subunit); CD132 (Common γ chain, IL-2Rγ); CDw136 (MSP-R, RON,p158-ron); CDw137 (4-1BB, ILA); CD139; CD141 (Thrombomodulin,Fetomodulin); CD147 (Basigin, EMMPRIN, M6, OX47); CD148 (HPTP-η, p260,DEP-1); CD155 (PVR); CD156a (CD156, ADAMS, MS2); CD156b (TACE, ADAM17,cSVP); CDw156C (ADAM10); CD157 (Mo5, BST-1); CD162 (PSGL-1); CD164(MGC-24, MUC-24); CD165 (AD2, gp37); CD168 (RHAMM, 1HABP, HMMR); CD169(Sialoadhesin, Siglec-1); CD170 (Siglec 5); CD171 (L1CAM, NILE); CD172(SIRP-1α, MyD-1); CD172b (SIRPβ); CD180 (RP105, Bgp95, Ly64); CD181(CXCR1, (Formerly known as CD128a)); CD182 (CXCR2, (Formerly known asCD128b)); CD184 (CXCR4, NPY3R); CD191 (CCR1); CD192 (CCR2); CD195(CCR5); CDw197 (CCR7 (was CDw197)); CDw198 (CCR8); CD204 (MSR); CD205(DEC-25); CD206 (MMR); CD207 (Langerin); CDw210 (CK); CD213a (CK);CDw217 (CK); CD220 (Insulin R); CD221 (IGF1R); CD222 (M6P-R, IGFII-R);CD224 (GGT); CD226 (DNAM-1, PTA1); CD230 (Prion Protein (PrP)); CD232(VESP-R); CD244 (2B4, P38, NAIL); CD245 (p220/240); CD256 (APRIL, TALL2,TNF (ligand) superfamily, member 13); CD257 (BLYS, TALL1, TNF (ligand)superfamily, member 13b); CD261 (TRAIL-R1, TNF-R superfamily, member10a); CD262 (TRAIL-R2, TNF-R superfamily, member 10b); CD263 (TRAIL-R3,TNBF-R superfamily, member 10c); CD264 (TRAIL-R4, TNF-R superfamily,member 10d); CD265 (TRANCE-R, TNF-R superfamily, member 11a); CD277(BT3.1, B7 family: Butyrophilin 3); CD280 (TEM22, ENDO180); CD281 (TLR1,TOLL-like receptor 1); CD282 (TLR2, TOLL-like receptor 2); CD284 (TLR4,TOLL-like receptor 4); CD295 (LEPR); CD298 (ATP1B3, Na K ATPase, β3subunit); CD300a (CMRF-35H); CD300c (CMRF-35A); CD300e (CMRF-35L1);CD302 (DCL1); CD305 (LAIR1); CD312 (EMR2); CD315 (CD9P1); CD317 (BST2);CD321 (JAM1); CD322 (JAM2); CDw328 (Siglec7); CDw329 (Siglec9); CD68 (gp110, Macrosialin); and/or mannose receptor; wherein the names listed inparentheses represent alternative names. Examples of such markersinclude those provided elsewhere herein.

In some embodiments, SCS-Mph targeting can be accomplished by anytargeting moiety that specifically binds to any entity (e.g., protein,lipid, carbohydrate, small molecule, etc.) that is prominently expressedand/or present on macrophages upon activation (i.e., activated SCS-Mphmarker). Exemplary activated SCS-Mph markers include, but are notlimited to, CD1a (R4, T6, HTA-1); CD1b (R1); CD1c (M241, R7); CD44R(CD44v, CD44v9); CD49d (VLA-4α, α4 Integrin); CD69 (AIM, EA 1, MLR3,34/28, VEA); CD105 (Endoglin); CD142 (Tissue factor, Thromboplastin,F3); CD143 (ACE, Peptidyl dipeptidase A, Kininase II); CD153 (CD30L,TNSF8); CD163 (M130, GHI/61, RM3/1); CD166 (ALCAM, KG-CAM, SC-1, BEN,DM-GRASP); CD227 (MUC1, PUM, PEM, EMA); CD253 (TRAIL, TNF (ligand)superfamily, member 10); CD273 (B7DC, PDL2); CD274 (B7H1,PDL1); CD275(B7H2, ICOSL); CD276 (B7H3); CD297 (ART4, ADP-ribosyltransferase 4; andDombrock blood group glycoprotein; wherein the names listed inparentheses represent alternative names. Examples of such markersinclude those provided elsewhere herein.

B Cell Targeting Moieties

In some embodiments, B cell targeting can be accomplished by moietiesthat bind the complement receptors, CR1 (i.e., CD35) or CR2 (i.e.,CD21), proteins which are expressed on B cells as well as FDCs. In someembodiments, B cell targeting can be accomplished by B cell markers suchas CD19, CD20, and/or CD22. In some embodiments, B cell targeting can beaccomplished by B cell markers such as CD40, CD52, CD80, CXCR5, VLA-4,class II MHC, surface IgM or IgD, APRL, and/or BAFF-R. The presentinvention encompasses the recognition that simultaneous targeting of Bcells by moieties specific for complement receptors or otherAPC-associated molecules boosts humoral responses.

In some embodiments, B cell targeting can be accomplished by anytargeting moiety that specifically binds to any entity (e.g., protein,lipid, carbohydrate, small molecule, etc.) that is prominently expressedand/or present on B cells (i.e., B cell marker). Exemplary B cellmarkers include, but are not limited to, CD1c (M241, R7); CD1d (R3); CD2(E-rosette R, T11, LFA-2); CD5 (T1, Tp67, Leu-1, Ly-1); CD6 (T12); CD9(p24, DRAP-1, MRP-1); CD11a (LFA-1α, αL Integrin chain); CD11b (αMIntegrin chain, CR3, Mo1, C3niR, Mac-1); CD11c (αX Integrin, P150, 95,AXb2); CDw17 (Lactosylceramide, LacCer); CD18 (Integrin β2, CD11a, b, cβ-subunit); CD19 (B4); CD20 (B1, Bp35); CD21 (CR2, EBV-R, C3dR); CD22(BL-CAM, Lyb8, Siglec-2); CD23 (FceRII, B6, BLAST-2, Leu-20); CD24(BBA-1, HSA); CD25 (Tac antigen, IL-2Rα, p55); CD26 (DPP IV ectoeneyme,ADA binding protein); CD27 (T14, S152); CD29 (Platelet GPIIa, β-1integrin, GP); CD31 (PECAM-1, Endocam); CD32 (FCγRII); CD35 (CR1,C3b/C4b receptor); CD37 (gp52-40); CD38 (ADP-ribosyl cyclase, T10); CD39(ATPdehydrogenase, NTPdehydrogenase-1); CD40 (Bp50); CD44 (ECMRII,H-CAM, Pgp-1); CD45 (LCA, T200, B220, Ly5); CD45RA; CD45RB; CD45RC;CD45RO (UCHL-1); CD46 (MCP); CD47 (gp42, IAP, OA3, Neurophilin); CD47R(MEM-133); CD48 (Blast-1, Hulym3, BCM-1, OX-45); CD49b (VLA-2α, gp1a, α2Integrin); CD49c (VLA-3α, α3 Integrin); CD49d (VLA-4α, α4 Integrin);CD50 (ICAM-3); CD52 (CAMPATH-1, HES); CD53 (OX-44); CD54 (ICAM-1); CD55(DAF); CD58 (LFA-3); CD60a (GD3); CD62L (L-selectin, LAM-1, LECAM-1,MEL-14, Leu8, TQ1); CD72 (Ly-19.2, Ly-32.2, Lyb-2); CD73(Ecto-5′-nuclotidase); CD74 (Ii, invariant chain); CD75 (sialo-maskedLactosamine); CD75S (α2, 6 sialytated Lactosamine); CD77 (Pk antigen,BLA, CTH/Gb3); CD79a (Igα, MB1); CD79b (Igβ, B29); CD80; CD81 (TAPA-1);CD82 (4F9, C33, IA4, KAI1 R2); CD83 (HB15); CD84 (P75, GR6); CD85j(ILT2, LIR1, MIR7); CDw92 (p70); CD95 (APO-1, FAS, TNFRSF6); CD98 (4F2,FRP-1, RL-388); CD99 (MIC2, E2); CD100 (SEMA4D); CD102 (ICAM-2); CD108(SEMA7A, JMH blood group antigen); CDw119 (IFNγR, IFNγRa); CD120a(TNFRI, p55); CD120b (TNFRII, p75, TNFR p80); CD121b (Type 2 IL-1R);CD122 (IL2Rβ); CD124 (IL-4Rα); CD130 (gp130); CD132 (Common γ chain,IL-2Rγ); CDw137 (4-1BB, ILA); CD139; CD147 (Basigin, EMMPRIN, M6, OX47);CD150 (SLAM, IPO-3); CD162 (PSGL-1); CD164 (MGC-24, MUC-24); CD166(ALCAM, KG-CAM, SC-1, BEN, DM-GRASP); CD167a (DDR1, trkE, cak); CD171(L1CMA, NILE); CD175s (Sialyl-Tn (S-Tn)); CD180 (RP105, Bgp95, Ly64);CD184 (CXCR4, NPY3R); CD185 (CXCR5); CD192 (CCR2); CD196 (CCR6); CD197(CCR7 (was CDw197)); CDw197 (CCR7, EBI1 BLR2); CD200 (OX2); CD205(DEC-205); CDw210 (CK); CD213a (CK); CDw217 (CK); CDw218a (IL18Rα);CDw218b (IL18Rβ); CD220 (Insulin R); CD221 (IGF1R); CD222 (M6P-R,IGFII-R); CD224 (GGT); CD225 (Leu13); CD226 (DNAM-1, PTA1); CD227 (MUC1,PUM, PEM, EMA); CD229 (Ly9); CD230 (Prion Protein (Prp)); CD232(VESP-R); CD245 (p220/240); CD247 (CD3 Zeta Chain); CD261 (TRAIL-R1,TNF-R superfamily, member 10a); CD262 (TRAIL-R2, TNF-R superfamily,member 10b); CD263 (TRAIL-R3, TNF-R superfamily, member 10c); CD264(TRAIL-R4, TNF-R superfamily, member 10d); CD265 (TRANCE-R, TNF-Rsuperfamily, member 11a); CD267 (TACI, TNF-R superfamily, member 13B);CD268 (BAFFR, TNF-R superfamily, member 13C); CD269 (BCMA, TNF-Rsuperfamily, member 16); CD275 (B7H2, ICOSL); CD277 (BT3.1.B7 family:Butyrophilin 3); CD295 (LEPR); CD298 (ATP1B3 Na K ATPase β3 subunit);CD300a (CMRF-35H); CD300c (CMRF-35A); CD305 (LAIR1); CD307 (IRTA2);CD315 (CD9P1); CD316 (EW12); CD317 (BST2); CD319 (CRACC, SLAMF7); CD321(JAM1); CD322 (JAM2); CDw327 (Siglec6, CD33L); CD68 (gp 100,Macrosialin); CXCR5; VLA-4; class II MHC; surface IgM; surface IgD;APRL; and/or BAFF-R; wherein the names listed in parentheses representalternative names. Examples of markers include those provided elsewhereherein.

In some embodiments, B cell targeting can be accomplished by anytargeting moiety that specifically binds to any entity (e.g., protein,lipid, carbohydrate, small molecule, etc.) that is prominently expressedand/or present on B cells upon activation (i.e., activated B cellmarker). Exemplary activated B cell markers include, but are not limitedto, CD1a (R4, T6, HTA-1); CD1b (R1); CD15s (Sialyl Lewis X); CD15u (3′sulpho Lewis X); CD15su (6 sulpho-sialyl Lewis X); CD30 (Ber-H2, Ki-1);CD69 (AIM, EA 1, MLR3, gp34/28, VEA); CD70 (Ki-24, CD27 ligand); CD80(B7, B7-1, BB1); CD86 (B7-2/B70); CD97 (BL-KDD/F12); CD125 (IL-5Rα);CD126 (IL-6Rα); CD138 (Syndecan-1, Heparan sulfate proteoglycan); CD152(CTLA-4); CD252 (OX40L, TNF(ligand) superfamily, member 4); CD253(TRAIL, TNF(ligand) superfamily, member 10); CD279 (PD1); CD289 (TLR9,TOLL-like receptor 9); and CD312 (EMR2); wherein the names listed inparentheses represent alternative names. Examples of markers includethose provided elsewhere herein.

Follicular Dendritic Cells

B cells that initially detect a previously unknown antigen typicallyexpress a B cell receptor (BCR, i.e., an antibody with a transmembranedomain) with suboptimal binding affinity for that antigen. However, Bcells can increase by several orders of magnitude the affinity of theantibodies they make when they enter into a germinal center (GC)reaction. This event, which typically lasts several weeks, depends onFDC that accumulate, retain and present antigenic material to theactivated B cells. B cells, while proliferating vigorously, repeatedlymutate the genomic sequences that encode the antigen binding site oftheir antibody and undergo class-switch recombination to form secretedhigh-affinity antibodies, mostly of the IgG isotype. GC reactions alsostimulate the generation of long-lived memory B cells and plasma cellsthat maintain high protective antibody titers, often for many years.Vaccine carriers that target FDC upon subcutaneous injection and thatare retained on the FDC surface for long periods of time are predictedto boost GC reactions in response to vaccination and improve theaffinity and longevity of desired humoral immune responses.

In some embodiments, FDC targeting can be accomplished by moieties thatbind the complement receptors, CR1 (i.e., CD35) or CR2 (i.e., CD21),proteins which are expressed on FDCs as well as B cells. Examples ofmoieties include those provided elsewhere herein.

Vaccine Nanocarriers Comprising Multiple Targeting Moieties

GC reactions and B cell survival not only require FDC, but also aredependent on help provided by activated CD4 T cells. Help is mostefficiently provided when a CD4 T cell is first stimulated by a DC thatpresents a cognate peptide in MHC class II (pMHC) to achieve afollicular helper (T_(FH)) phenotype. The newly generated T_(FH) cellthen migrates toward the B follicle and provides help to those B cellsthat present them with the same pMHC complex. For this, B cells firstacquire antigenic material (e.g., virus or virus-like vaccine),internalize and process it (i.e., extract peptide that is loaded intoMHC class II), and then present the pMHC to a T_(FH) cell.

Thus, the present invention encompasses the recognition that a vaccinethat stimulates optimal humoral immunity can combine several featuresand components (FIG. 1): (a) antigenic material for CD4 T cells that istargeted to and presented by DCs; (b) high density surface antigens thatcan be presented in their native form by SCS-Mph to antigen-specificfollicular B cells; (c) the capacity to be acquired and processed byfollicular B cells for presentation to T_(FH) cells (the presentinvention encompasses the recognition that B cells readily acquire andinternalize particulate matter from SCS-Mph); (d) the ability to reachFDC and be retained on FDC in intact form and for long periods of time;and (e) adjuvant activity to render APC fully immunogenic and to avoidor overcome tolerance.

In some embodiments, a vaccine nanocarrier comprises at least onetargeting moiety. In some embodiments, all of the targeting moieties ofa vaccine nanocarrier are identical to one another. In some embodiments,a vaccine nanocarrier a number of different types of targeting moieties.In some embodiments, a vaccine nanocarrier comprises multiple individualtargeting moieties, all of which are identical to one another. In someembodiments, a vaccine nanocarrier comprises exactly one type oftargeting moiety. In some embodiments, a vaccine nanocarrier comprisesexactly two distinct types of targeting moieties. In some embodiments, avaccine nanocarrier comprises greater than two distinct types oftargeting moieties.

In some embodiments, a vaccine nanocarrier comprises a single type oftargeting moiety that directs delivery of the vaccine nanocarrier to asingle cell type (e.g., delivery to SCS-Mph only). In some embodiments,a vaccine nanocarrier comprises a single type of targeting moiety thatdirects delivery of the vaccine nanocarrier to multiple cell types(e.g., delivery to both SCS-Mph and FDCs). In some embodiments, avaccine nanocarrier comprises two types of targeting moieties, whereinthe first type of targeting moiety directs delivery of the vaccinenanocarrier to one cell type, and the second type of targeting moietydirects delivery of the vaccine nanocarrier to a second cell type. Insome embodiments, a vaccine nanocarrier comprises greater than two typesof targeting moieties, wherein one or more types of targeting moietiesdirect delivery of the vaccine nanocarrier to one cell type, and one ormore types of targeting moieties direct delivery of the vaccinenanocarrier to a second cell type. To give but one example, a vaccinenanocarrier may comprise two types of targeting moieties, wherein thefirst type of targeting moiety directs delivery of the vaccinenanocarrier to DCs, and the second type of targeting moiety directsdelivery of the vaccine nanocarrier to SCS-Mph.

In some embodiments, a vaccine nanocarrier comprises at least onetargeting moiety that is associated with the exterior surface of thevaccine nanocarrier. In some embodiments, the association is covalent.In some embodiments, the covalent association is mediated by one or morelinkers. In some embodiments, the association is non-covalent. In someembodiments, the non-covalent association is mediated by chargeinteractions, affinity interactions, metal coordination, physicaladsorption, host-guest interactions, hydrophobic interactions, TTstacking interactions, hydrogen bonding interactions, van der Waalsinteractions, magnetic interactions, electrostatic interactions,dipole-dipole interactions, and/or combinations thereof.

In some embodiments, a vaccine nanocarrier comprises a lipid membrane(e.g., lipid bilayer, lipid monolayer, etc.), wherein at least onetargeting moiety is associated with the lipid membrane. In someembodiments, at least one targeting moiety is embedded within the lipidmembrane. In some embodiments, at least one targeting moiety is embeddedwithin the lumen of a lipid bilayer. In some embodiments, at least onetargeting moiety may be located at multiple locations of a vaccinenanocarrier. For example, a first targeting moiety may be embeddedwithin a lipid membrane, and a second immunostimulatory agent may beassociated with the exterior surface of a vaccine nanocarrier. To giveanother example, a first targeting moiety and a second targeting moietymay both be associated with the exterior surface of a vaccinenanocarrier.

Immunostimulatory Agents

As described above, in some embodiments, vaccine nanocarriers maytransport one or more immunostimulatory agents which can help stimulateimmune responses. In some embodiments, a vaccine nanocarrier comprises asingle type of immunostimulatory agent that stimulates both B cells andT cells. In some embodiments, a vaccine nanocarrier comprises two typesof immunostimulatory agents, wherein first type of immunostimulatoryagent stimulates B cells, and the second type of immunostimulatory agentstimulates T cells. In some embodiments, a vaccine nanocarrier comprisesgreater than two types of immunostimulatory agents, wherein one or moretypes of immunostimulatory agents stimulate B cells, and one or moretypes of immunostimulatory agents stimulate T cells. See the sectionabove for a more detailed description of immunostimulatory agents thatcan be used in accordance with the present invention.

Assays for B Cell Activation

In some embodiments, various assays can be utilized in order todetermine whether an immune response has been stimulated in a B cell orgroup of B cells (i.e., whether a B cell or group of B cells has become“activated”). In some embodiments, stimulation of an immune response inB cells can be determined by measuring antibody titers. In general,“antibody titer” refers to the ability of antibodies to bind andneutralize antigens at particular dilutions. For example, a highantibody titer refers to the ability of antibodies to bind andneutralize antigens even at high dilutions. In some embodiments, animmune response in B cells is said to be stimulated if antibody titersare measured to be positive at dilutions at least about 5-fold greater,at least about 10-fold greater, at least about 20-fold greater, at leastabout 50-fold greater, at least about 100-fold greater, at least about500-fold greater, at least about 1000 fold greater, or more than about1000-fold greater than in non-immunized individuals or pre-immune serum.

In some embodiments, stimulation of an immune response in B cells can bedetermined by measuring antibody affinity. In particular, an immuneresponse in B cells is said to be stimulated if an antibody has anequilibrium dissociation constant (K_(d)) less than 10⁻⁷ M, less than10⁻⁸ M, less than 10⁻⁹ M, less than 10⁻¹⁰ M, less than 10⁻¹¹ M, lessthan 10⁻¹² M, or less.

In some embodiments, a T cell-dependent immune response in B cells issaid to be stimulated if class-switch recombination has occurred. Inparticular, a switch from IgM to an IgG isotype or to IgA or to amixture of these isotypes is indicative of a T cell dependent immuneresponse in B cells.

In some embodiments, an immune response in B cells is determined bymeasuring affinity maturation of antigen-specific antibodies. Affinitymaturation occurs during the germinal center reaction whereby activatedB cells repeatedly mutate a region of the immunoglobulin gene thatencodes the antigen-binding region. B cells producing mutated antibodieswhich have a higher affinity for antigen are preferentially allowed tosurvive and proliferate. Thus, over time, the antibodies made by B cellsin GCs acquire incrementally higher affinities. In some embodiments, thereadout of this process is the presence of high antibody titer (e.g.high affinity IgG antibodies that bind and neutralize antigens even athigh dilutions).

In some embodiments, an immune response in B cells is said to bestimulated if memory B cells and/or long-lived plasma cells that canproduce large amounts of high-affinity antibodies for extended periodsof time have formed. In some embodiments, antibody titers are measuredafter different time intervals (e.g. 2 weeks, 1 month, 2 months, 6months, 1 year, 2 years, 5 years, 10 years, 15 years, 20 years, 25years, or longer) after vaccination in order to test for the presence ofmemory B cells and/or long-lived plasma cells that can produce largeamounts of high-affinity antibodies for extended periods of time. Insome embodiments, memory B cells and/or long-lived plasma cells that canproduce large amounts of high-affinity antibodies for extended periodsof time are said to be present by measuring humoral responses (e.g., ifhumoral responses are markedly more rapid and result in higher titersafter a later booster vaccination than during the initialsensitization).

In some embodiments, an immune response in B cells is said to bestimulated if a vigorous germinal center reaction occurs. In someembodiments, a vigorous germinal center reaction can be assessedvisually by performing histology experiments. In some embodiments,vigorous germinal center reaction can be assayed by performingimmunohistochemistry of antigen-containing lymphoid tissues (e.g.,vaccine-draining lymph nodes, spleen, etc.). In some embodiments,immunohistochemistry is followed by flow cytometry.

In some embodiments, stimulation of an immune response in B cells can bedetermined by identifying antibody isotypes (e.g., IgG, IgA, IgE, IgM).In certain embodiments, production of IgG isotype antibodies by B cellsis a desirable immune response in a B cell.

In some embodiments, an immune response in B cells is determined byanalyzing antibody function in neutralization assays. In particular, theability of a microorganism (e.g., virus, bacterium, fungus, protozoan,parasite, etc.) to infect a susceptible cell line in vitro in theabsence of serum is compared to conditions when different dilutions ofimmune and non-immune serum are added to the culture medium in which thecells are grown. In certain embodiments, an immune response in a B cellis said to be stimulated if infection of a microorganism is neutralizedat a dilution of about 1:5, about 1:10, about 1:50, about 1:100, about1:500, about 1:1000, about 1:5000, about 1:10,000, or less.

In some embodiments, the efficacy of vaccines in animal models may bedetermined by infecting groups of immunized and non-immunized mice(e.g., 3 or more weeks after vaccination) with a dose of a microorganismthat is typically lethal. The magnitude and duration of survival of bothgroup is monitored and typically graphed a Kaplan-Meier curves. Toassess whether enhanced survival is due to B cell responses, serum fromimmune mice can be transferred as a “passive vaccine” to assessprotection of non-immune mice from lethal infection.

One of ordinary skill in the art will recognize that the assaysdescribed above are only exemplary methods which could be utilized inorder to determine whether B cell activation has occurred. Any assayknown to one of skill in the art which can be used to determine whetherB cell activation has occurred falls within the scope of this invention.The assays described herein as well as additional assays that could beused to determine whether B cell activation has occurred are describedin Current Protocols in Immunology (John Wiley & Sons, Hoboken, N.Y.,2007; incorporated herein by reference).

Nanocarriers

Any of the nanocarriers described herein may be vaccine nanocarriers.Although some of disclosure provided herein specifically mentionsvaccine nanocarriers, it will be appreciated that, unless otherwisespecified, the disclosure is not limited to vaccine nanocarriers butapplies to any of the nanocarriers described herein.

In some embodiments, a vaccine nanocarrier is a synthetic nanocarrierthat comprises, for example, at least one immunomodulatory agent whichis capable of stimulating an immune response in one or both of B cellsand T cells. The immunomodulatory agent may be associated with thenanocarriers in any way as described in more detail herein.

In some embodiments, nanocarriers are biodegradable and biocompatible.In general, a biocompatible substance is not toxic to cells. In someembodiments, a substance is considered to be biocompatible if itsaddition to cells results in less than a certain threshold of cell death(e.g. less than 50%, 20%, 10%, 5%, or less cell death). In someembodiments, a substance is considered to be biocompatible if itsaddition to cells does not induce adverse effects. In general, abiodegradable substance is one that undergoes breakdown underphysiological conditions over the course of a therapeutically relevanttime period (e.g., weeks, months, or years). In some embodiments, abiodegradable substance is a substance that can be broken down bycellular machinery. In some embodiments, a biodegradable substance is asubstance that can be broken down by chemical processes. In someembodiments, a nanocarrier is a substance that is both biocompatible andbiodegradable. In some embodiments, a nanocarrier is a substance that isbiocompatible, but not biodegradable. In some embodiments, a nanocarrieris a substance that is biodegradable, but not biocompatible.

In some embodiments, a nanocarrier in accordance with the presentinvention is any entity having a greatest dimension (e.g., diameter) ofless than 5 microns (μm). In some embodiments, inventive nanocarriershave a greatest dimension of less than 3 μm. In some embodiments,inventive nanocarriers have a greatest dimension of less than 1000nanometers (nm). In some embodiments, inventive nanocarriers have agreatest dimension of less than 900 nm, 800 nm, 700 nm, 600 nm, 500 nm,400 nm, 300 nm, 200 nm, or 100 nm. In some embodiments, inventivenanocarriers have a greatest dimension (e.g., diameter) of 300 nm orless. In some embodiments, inventive nanocarriers have a greatestdimension (e.g., diameter) of 250 nm or less. In some embodiments,inventive nanocarriers have a greatest dimension (e.g., diameter) of 200nm or less. In some embodiments, inventive nanocarriers have a greatestdimension (e.g., diameter) of 150 nm or less. In some embodiments,inventive nanocarriers have a greatest dimension (e.g., diameter) of 100nm or less. Smaller nanocarriers, e.g., having a greatest dimension of50 nm or less are used in some embodiments of the invention. In someembodiments, inventive nanocarriers have a greatest dimension rangingbetween 25 nm and 200 nm. In some embodiments, inventive nanocarriershave a greatest dimension ranging between 20 nm and 100 nm. Thenanocarriers of the compositions provided herein, in some embodiments,have a mean geometric diameter that is less than 500 nm. In someembodiments, the nanocarriers have mean geometric diameter that isgreater than 50 nm but less than 500 nm. In some embodiments, the meangeometric diameter of a population of nanocarriers is about 60 nm, 75nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, or 475 nm. In someembodiments, the mean geometric diameter is between 100-400 nm, 100-300nm, 100-250 nm, or 100-200 nm. In some embodiments, the mean geometricdiameter is between 60-400 nm, 60-350 nm, 60-300 nm, 60-250 nm, or60-200 nm. In some embodiments, the mean geometric diameter is between75-250 nm. In some embodiments, inventive nanocarriers have a greatestdimension of greater than 1000 nanometers (nm). In some embodiments,inventive nanocarriers have a greatest dimension of greater than 900 nm,800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm. Insome embodiments, inventive nanocarriers have a greatest dimension(e.g., diameter) of 300 nm or more. In some embodiments, inventivenanocarriers have a greatest dimension (e.g., diameter) of 250 nm ormore. In some embodiments, inventive nanocarriers have a greatestdimension (e.g., diameter) of 200 nm or more. In some embodiments,inventive nanocarriers have a greatest dimension (e.g., diameter) of 150nm or more. In some embodiments, inventive nanocarriers have a greatestdimension (e.g., diameter) of 100 nm or more. Smaller nanocarriers,e.g., having a greatest dimension of 50 nm or more are used in someembodiments of the invention.

In some embodiments, nanocarriers have a diameter of less than 1000 nm.In some embodiments, nanocarriers have a diameter of approximately 750nm. In some embodiments, nanocarriers have a diameter of approximately500 nm. In some embodiments, nanocarriers have a diameter ofapproximately 450 nm. In some embodiments, nanocarriers have a diameterof approximately 400 nm. In some embodiments, nanocarriers have adiameter of approximately 350 nm. In some embodiments, nanocarriers havea diameter of approximately 300 nm. In some embodiments, nanocarriershave a diameter of approximately 275 nm. In some embodiments,nanocarriers have a diameter of approximately 250 nm. In someembodiments, nanocarriers have a diameter of approximately 225 nm. Insome embodiments, nanocarriers have a diameter of approximately 200 nm.In some embodiments, nanocarriers have a diameter of approximately 175nm. In some embodiments, nanocarriers have a diameter of approximately150 nm. In some embodiments, nanocarriers have a diameter ofapproximately 125 nm. In some embodiments, nanocarriers have a diameterof approximately 100 nm. In some embodiments, nanocarriers have adiameter of approximately 75 nm. In some embodiments, nanocarriers havea diameter of approximately 50 nm. In some embodiments, nanocarriershave a diameter of approximately 25 nm.

In certain embodiments, nanocarriers are greater in size than the renalexcretion limit (e.g., nanocarriers having diameters of greater than 6nm). In certain embodiments, nanocarriers are small enough to avoidclearance of nanocarriers from the bloodstream by the liver (e.g.,nanocarriers having diameters of less than 1000 nm). In general,physiochemical features of nanocarriers should allow a nanocarrier tocirculate longer in plasma by decreasing renal excretion and liverclearance.

It is often desirable to use a population of nanocarriers that isrelatively uniform in terms of size, shape, and/or composition so thateach nanocarrier has similar properties. For example, at least 80%, atleast 90%, or at least 95% of the nanocarriers may have a diameter orgreatest dimension that falls within 5%, 10%, or 20% of the averagediameter or greatest dimension. In some embodiments, a population ofnanocarriers may be heterogeneous with respect to size, shape, and/orcomposition. In some embodiments, 30%, 40%, 50%, 60%, 70%, 80%, 90%, ormore of the nanocarriers of a population of nanocarriers have a diameterthat is less than 500 nM. In some embodiments, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or more of the nanocarriers of a population ofnanocarriers have a diameter that is greater than 50 nm but less than500 nm. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or more of the nanocarriers of a population of nanocarriers have adiameter of about 60 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm,225 nm, 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm,450 nm, or 475 nm. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or more of the nanocarriers of a population ofnanocarriers have a diameter that is between 100-400 nm, 100-300 nm,100-250 nm, or 100-200 nm. In some embodiments, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or more of the nanocarriers of a population ofnanocarriers have a diameter that is between 60-400 nm, 60-350 nm,60-300 nm, 60-250 nm, or 60-200 nm.

A variety of different nanocarriers can be used in accordance with thepresent invention. In some embodiments, nanocarriers are spheres orspheroids. In some embodiments, nanocarriers are flat or plate-shaped.In some embodiments, nanocarriers are cubes or cuboids. In someembodiments, nanocarriers are ovals or ellipses. In some embodiments,nanocarriers are cylinders, cones, toroids (i.e., donut shaped), orpyramids. In some embodiments, particles are liposomes. In someembodiments, particles are micelles. It will be appreciated that each ofthese shapes fall within the general category of “particles,” and thatthe nanocarriers of the invention may comprise nanoparticles (i.e., aparticle having a diameter of less than 1000 nm), microparticles (i.e.,particles having a diameter of less than 1000 micrometers), orpicoparticles (i.e., particles having a diameter of less than 1 nm). Itwill further be appreciated that, in some embodiments (such aslipid-based nanocarriers, as described in more detail below), thenanocarriers are not rigid and may have a shape and diameter thatchanges based on the environment.

“Maximum dimension of a synthetic nanocarrier” means the largestdimension of a nanocarrier measured along any axis of the syntheticnanocarrier. “Minimum dimension of a synthetic nanocarrier” means thesmallest dimension of a synthetic nanocarrier measured along any axis ofthe synthetic nanocarrier. For example, for a spheriodal syntheticnanocarrier, the maximum and minimum dimension of a syntheticnanocarrier would be substantially identical, and would be the size ofits diameter. Similarly, for a cubic synthetic nanocarrier, the minimumdimension of a synthetic nanocarrier would be the smallest of itsheight, width or length, while the maximum dimension of a syntheticnanocarrier would be the largest of its height, width or length. In anembodiment, a minimum dimension of at least 75%, preferably 80%, morepreferably 90%, of the synthetic nanocarriers in a sample, based on thetotal number of synthetic nanocarriers in the sample, is greater than100 nm. In a embodiment, a maximum dimension of at least 75%, preferably80%, more preferably 90%, of the synthetic nanocarriers in a sample,based on the total number of synthetic nanocarriers in the sample, isequal to or less than 5 μm. Preferably, a minimum dimension of at least75%, preferably 80%, more preferably 90%, of the synthetic nanocarriersin a sample, based on the total number of synthetic nanocarriers in thesample, is greater than 110 nm, more preferably greater than 120 nm,more preferably greater than 130 nm, and more preferably still greaterthan 150 nm. Preferably, a maximum dimension of at least 75%, preferably80%, more preferably 90%, of the synthetic nanocarriers in a sample,based on the total number of synthetic nanocarriers in the sample isequal to or less than 3 μm, more preferably equal to or less than 2 μm,more preferably equal to or less than 1 μm, more preferably equal to orless than 800 nm, more preferably equal to or less than 600 nm, and morepreferably still equal to or less than 500 nm. In preferred embodiments,a maximum dimension of at least 75%, preferably 80%, more preferably90%, of the synthetic nanocarriers in a sample, based on the totalnumber of synthetic nanocarriers in the sample, is equal to or greaterthan 100 nm, more preferably equal to or greater than 120, morepreferably greater than 130 nm, more preferably greater than 140 nm, andmore preferably still greater than 150 nm. Measurement of syntheticnanocarrier sizes is obtained by suspending the synthetic nanocarriersin a liquid (usually aqueous) media and using dynamic light scattering(e.g. using a Brookhaven ZetaPALS instrument)Nanocarriers can be solidor hollow and can comprise one or more layers. In some embodiments, eachlayer has a unique composition and unique properties relative to theother layer(s). To give but one example, nanocarriers may have acore/shell structure, wherein the core is one layer (e.g. a polymericcore) and the shell is a second layer (e.g. a lipid bilayer ormonolayer). Nanocarriers may comprise a plurality of different layers.In some embodiments, one layer may be substantially cross-linked, asecond layer is not substantially cross-linked, and so forth. In someembodiments, one, a few, or all of the different layers may comprise oneor more immunomodulatory agents, targeting moieties, immunostimulatoryagents, and/or combinations thereof. In some embodiments, one layercomprises an immunomodulatory agent, targeting moiety, and/orimmunostimulatory agent, a second layer does not comprise animmunomodulatory agent, targeting moiety, and/or immunostimulatoryagent, and so forth. In some embodiments, each individual layercomprises a different immunomodulatory agent, targeting moiety,immunostimulatory agent, and/or combination thereof.

Lipid Nanocarriers

In some embodiments, nanocarriers may optionally comprise one or morelipids. In some embodiments, a nanocarrier may comprise a liposome. Insome embodiments, a nanocarrier may comprise a lipid bilayer. In someembodiments, a nanocarrier may comprise a lipid monolayer. In someembodiments, a nanocarrier may comprise a micelle. In some embodiments,a nanocarrier may comprise a core comprising a polymeric matrixsurrounded by a lipid layer (e.g., lipid bilayer, lipid monolayer,etc.). In some embodiments, a nanocarrier may comprise a non-polymericcore (e.g., metal particle, quantum dot, ceramic particle, boneparticle, viral particle, etc.) surrounded by a lipid layer (e.g., lipidbilayer, lipid monolayer, etc.).

In some embodiments, nanocarriers may comprise a lipid bilayer orientedsuch that the interior and exterior of the nanocarrier are hydrophilic,and the lumen of the lipid bilayer is hydrophobic. Examples of vaccinenanocarriers comprising lipid bilayers are described in Example 2 andshown in FIGS. 3-8. In some embodiments, hydrophobic immunomodulatoryagents, targeting moieties, and/or immunostimulatory agents may beassociated with (e.g., embedded within) the lumen of the lipid bilayer.In some embodiments, hydrophilic immunomodulatory agents, targetingmoieties, and/or immunostimulatory agents may be associated with (e.g.,covalently or non-covalently associated with, encapsulated within, etc.)the interior and/or exterior of the nanocarrier. In some embodiments,hydrophilic immunomodulatory agents, targeting moieties, and/orimmunostimulatory agents may be associated with (e.g., covalently ornon-covalently associated with, encapsulated within, etc.) the interiorand/or exterior surface of the lipid bilayer. In some embodiments, theinterior, hydrophilic surface of the lipid bilayer is associated with anamphiphilic entity. In some embodiments, the amphiphilic entity isoriented such that the hydrophilic end of the amphiphilic entity isassociated with the interior surface of the lipid bilayer, and thehydrophobic end of the amphiphilic entity is oriented toward theinterior of the nanocarrier, producing a hydrophobic environment withinthe nanocarrier interior.

In some embodiments, nanocarriers may comprise a lipid monolayeroriented such that the interior of the nanocarrier is hydrophobic, andthe exterior of the nanocarrier is hydrophilic. Examples of vaccinenanocarriers comprising lipid monolayers are described in Example 2 andshown in FIGS. 9 and 10. In some embodiments, hydrophobicimmunomodulatory agents, targeting moieties, and/or immunostimulatoryagents may be associated with (e.g., covalently or non-covalentlyassociated with, encapsulated within, etc.) the interior of thenanocarrier and/or the interior surface of the lipid monolayer. In someembodiments, hydrophilic immunomodulatory agents, targeting moieties,and/or immunostimulatory agents may be associated with (e.g., covalentlyor non-covalently associated with, encapsulated within, etc.) theexterior of the nanocarrier and/or the exterior surface of the lipidmonolayer. In some embodiments, the interior, hydrophobic surface of thelipid bilayer is associated with an amphiphilic entity. In someembodiments, the amphiphilic entity is oriented such that thehydrophobic end of the amphiphilic entity is associated with theinterior surface of the lipid bilayer, and the hydrophilic end of theamphiphilic entity is oriented toward the interior of the nanocarrier,producing a hydrophilic environment within the nanocarrier interior.

In some embodiments, a nanocarrier may comprise one or morenanoparticles associated with the exterior surface of the nanocarrier.Examples of vaccine nanocarriers comprising nanoparticles associatedwith the exterior surface of the nanocarrier are described in Example 2and shown in FIGS. 4, 6, and 8.

The percent of lipid in nanocarriers can range from 0% to 99% by weight,from 10% to 99% by weight, from 25% to 99% by weight, from 50% to 99% byweight, or from 75% to 99% by weight. In some embodiments, the percentof lipid in nanocarriers can range from 0% to 75% by weight, from 0% to50% by weight, from 0% to 25% by weight, or from 0% to 10% by weight. Insome embodiments, the percent of lipid in nanocarriers can beapproximately 1% by weight, approximately 2% by weight, approximately 3%by weight, approximately 4% by weight, approximately 5% by weight,approximately 10% by weight, approximately 15% by weight, approximately20% by weight, approximately 25% by weight, or approximately 30% byweight.

In some embodiments, lipids are oils. In general, any oil known in theart can be included in nanocarriers. In some embodiments, an oil maycomprise one or more fatty acid groups or salts thereof. In someembodiments, a fatty acid group may comprise digestible, long chain(e.g., C₈-C₅₀), substituted or unsubstituted hydrocarbons. In someembodiments, a fatty acid group may be a C₁₀-C₂₀ fatty acid or saltthereof. In some embodiments, a fatty acid group may be a C₁₅-C₂₀ fattyacid or salt thereof. In some embodiments, a fatty acid group may be aC₁₅-C₂₅ fatty acid or salt thereof. In some embodiments, a fatty acidgroup may be unsaturated. In some embodiments, a fatty acid group may bemonounsaturated. In some embodiments, a fatty acid group may bepolyunsaturated. In some embodiments, a double bond of an unsaturatedfatty acid group may be in the cis conformation. In some embodiments, adouble bond of an unsaturated fatty acid may be in the transconformation.

In some embodiments, a fatty acid group may be one or more of butyric,caproic, caprylic, capric, lauric, myristic, palmitic, stearic,arachidic, behenic, or lignoceric acid. In some embodiments, a fattyacid group may be one or more of palmitoleic, oleic, vaccenic, linoleic,alpha-linolenic, gamma-linoleic, arachidonic, gadoleic, arachidonic,eicosapentaenoic, docosahexaenoic, or erucic acid.

In some embodiments, the oil is a liquid triglyceride.

Suitable oils for use with the present invention include, but are notlimited to, almond, apricot kernel, avocado, babassu, bergamot, blackcurrent seed, borage, cade, camomile, canola, caraway, carnauba, castor,cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed,emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd,grape seed, hazel nut, hyssop, jojoba, kukui nut, lavandin, lavender,lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoamseed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel,peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran,rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn,sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle,tsubaki, vetiver, walnut, and wheat germ oils, and combinations thereof.Suitable oils for use with the present invention include, but are notlimited to, butyl stearate, caprylic triglyceride, capric triglyceride,cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate,mineral oil, octyldodecanol, oleyl alcohol, silicone oil, andcombinations thereof. In some embodiments, a lipid is a hormone (e.g.estrogen, testosterone), steroid (e.g., cholesterol, bile acid), vitamin(e.g. vitamin E), phospholipid (e.g. phosphatidyl choline), sphingolipid(e.g. ceramides), or lipoprotein (e.g. apolipoprotein).

Nanocarriers Comprising a Polymeric Matrix

In some embodiments, nanocarriers can comprise one or more polymers. Insome embodiments, a polymeric matrix can be surrounded by a coatinglayer (e.g., liposome, lipid monolayer, micelle, etc.). In someembodiments, an immunomodulatory agent, targeting moiety, and/orimmunostimulatory agent can be associated with the polymeric matrix. Insuch embodiments, the immunomodulatory agent, targeting moiety, and/orimmunostimulatory agent is effectively encapsulated within thenanocarrier.

In some embodiments, an immunomodulatory agent, targeting moiety, and/orimmunostimulatory agent can be covalently associated with a polymericmatrix. In some embodiments, covalent association is mediated by alinker. In some embodiments, an immunomodulatory agent, targetingmoiety, and/or immunostimulatory agent can be non-covalently associatedwith a polymeric matrix. For example, in some embodiments, animmunomodulatory agent, targeting moiety, and/or immunostimulatory agentcan be encapsulated within, surrounded by, and/or dispersed throughout apolymeric matrix. Alternatively or additionally, an immunomodulatoryagent, targeting moiety, and/or immunostimulatory agent can beassociated with a polymeric matrix by hydrophobic interactions, chargeinteractions, van der Waals forces, etc.

A wide variety of polymers and methods for forming polymeric matricestherefrom are known in the art of drug delivery. In general, a polymericmatrix comprises one or more polymers. Any polymer may be used inaccordance with the present invention. Polymers may be natural orunnatural (synthetic) polymers. Polymers may be homopolymers orcopolymers comprising two or more monomers. In terms of sequence,copolymers may be random, block, or comprise a combination of random andblock sequences. Typically, polymers in accordance with the presentinvention are organic polymers.

Examples of polymers include polyethylenes, polycarbonates (e.g.poly(1,3-dioxan-2one)), polyanhydrides (e.g. poly(sebacic anhydride)),polyhydroxyacids (e.g. poly(β-hydroxyalkanoate)), polypropylfumerates,polycaprolactones, polyamides (e.g. polycaprolactam), polyacetals,polyethers, polyesters (e.g., polylactide, polyglycolide),poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polyureas, polystyrenes, and polyamines.

In some embodiments, polymers in accordance with the present inventioninclude polymers which have been approved for use in humans by the U.S.Food and Drug Administration (FDA) under 21 C.F.R. §177.2600, includingbut not limited to polyesters (e.g., polylactic acid,poly(lactic-co-glycolic acid), polycaprolactone, polyvalerolactone,poly(1,3-dioxan-2one)); polyanhydrides (e.g., poly(sebacic anhydride));polyethers (e.g., polyethylene glycol); polyurethanes;polymethacrylates; polyacrylates; and polycyanoacrylates.

In some embodiments, polymers can be hydrophilic. For example, polymersmay comprise anionic groups (e.g., phosphate group, sulphate group,carboxylate group); cationic groups (e.g., quaternary amine group); orpolar groups (e.g., hydroxyl group, thiol group, amine group). In someembodiments, a nanocarrier comprising a hydrophilic polymeric matrixgenerates a hydrophilic environment within the nanocarrier. In someembodiments, hydrophilic immunomodulatory agents, targeting moieties,and/or immunostimulatory agents may be associated with hydrophilicpolymeric matrices.

In some embodiments, polymers can be hydrophobic. In some embodiments, ananocarrier comprising a hydrophobic polymeric matrix generates ahydrophobic environment within the nanocarrier. In some embodiments,hydrophobic immunomodulatory agents, targeting moieties, and/orimmunostimulatory agents may be associated with hydrophobic polymericmatrices.

In some embodiments, polymers may be modified with one or more moietiesand/or functional groups. Any moiety or functional group can be used inaccordance with the present invention. In some embodiments, polymers maybe modified with polyethylene glycol (PEG), with a carbohydrate, and/orwith acyclic polyacetals derived from polysaccharides (Papisov, 2001,ACS Symposium Series, 786:301; incorporated herein by reference).

In some embodiments, polymers may be modified with a lipid or fatty acidgroup, properties of which are described in further detail below. Insome embodiments, a fatty acid group may be one or more of butyric,caproic, caprylic, capric, lauric, myristic, palmitic, stearic,arachidic, behenic, or lignoceric acid. In some embodiments, a fattyacid group may be one or more of palmitoleic, oleic, vaccenic, linoleic,alpha-linoleic, gamma-linoleic, arachidonic, gadoleic, arachidonic,eicosapentaenoic, docosahexaenoic, or erucic acid.

In some embodiments, polymers may be polyesters, including copolymerscomprising lactic acid and glycolic acid units, such as poly(lacticacid-co-glycolic acid) and poly(lactide-co-glycolide), collectivelyreferred to herein as “PLGA”; and homopolymers comprising glycolic acidunits, referred to herein as “PGA,” and lactic acid units, such aspoly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid,poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectivelyreferred to herein as “PLA.” In some embodiments, exemplary polyestersinclude, for example, polyhydroxyacids; PEG copolymers and copolymers oflactide and glycolide (e.g., PLA-PEG copolymers, PGA-PEG copolymers,PLGA-PEG copolymers, and derivatives thereof. In some embodiments,polyesters include, for example, polyanhydrides, poly(ortho ester),poly(ortho ester)-PEG copolymers, poly(caprolactone),poly(caprolactone)-PEG copolymers, polylysine, polylysine-PEGcopolymers, poly(ethylene imine), poly(ethylene imine)-PEG copolymers,poly(L-lactide-co-L-lysine), poly(serine ester),poly(4-hydroxy-L-proline ester), poly[α-(4-aminobutyl)-L-glycolic acid],and derivatives thereof.

In some embodiments, a polymer may be PLGA. PLGA is a biocompatible andbiodegradable co-polymer of lactic acid and glycolic acid, and variousforms of PLGA are characterized by the ratio of lactic acid:glycolicacid. Lactic acid can be L-lactic acid, D-lactic acid, or D,L-lacticacid. The degradation rate of PLGA can be adjusted by altering thelactic acid:glycolic acid ratio. In some embodiments, PLGA to be used inaccordance with the present invention is characterized by a lacticacid:glycolic acid ratio of approximately 85:15, approximately 75:25,approximately 60:40, approximately 50:50, approximately 40:60,approximately 25:75, or approximately 15:85.

In some embodiments, polymers may be one or more acrylic polymers. Incertain embodiments, acrylic polymers include, for example, acrylic acidand methacrylic acid copolymers, methyl methacrylate copolymers,ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkylmethacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),methacrylic acid alkylamide copolymer, poly(methyl methacrylate),poly(methacrylic acid anhydride), methyl methacrylate, polymethacrylate,poly(methyl methacrylate) copolymer, polyacrylamide, aminoalkylmethacrylate copolymer, glycidyl methacrylate copolymers,polycyanoacrylates, and combinations comprising one or more of theforegoing polymers. The acrylic polymer may comprise fully-polymerizedcopolymers of acrylic and methacrylic acid esters with a low content ofquaternary ammonium groups.

In some embodiments, polymers can be cationic polymers. In general,cationic polymers are able to condense and/or protect negatively chargedstrands of nucleic acids (e.g. DNA, RNA, or derivatives thereof).Amine-containing polymers such as poly(lysine) (Zauner et al., 1998,Adv. Drug Del. Rev., 30:97; and Kabanov et al., 1995, BioconjugateChem., 6:7; both of which are incorporated herein by reference),poly(ethylene imine) (PEI; Boussif et al., 1995, Proc. Natl. Acad. Sci.,USA, 1995, 92:7297; incorporated herein by reference), andpoly(amidoamine) dendrimers (Kukowska-Latallo et al., 1996, Proc. Natl.Acad. Sci., USA, 93:4897; Tang et al., 1996, Bioconjugate Chem., 7:703;and Haensler et al., 1993, Bioconjugate Chem., 4:372; all of which areincorporated herein by reference) are positively-charged atphysiological pH, form ion pairs with nucleic acids, and mediatetransfection in a variety of cell lines.

In some embodiments, polymers can be degradable polyesters bearingcationic side chains (Putnam et al., 1999, Macromolecules, 32:3658;Barrera et al., 1993, J. Am. Chem. Soc., 115:11010; Kwon et al., 1989,Macromolecules, 22:3250; Lim et al., 1999, J. Am. Chem. Soc., 121:5633;and Zhou et al., 1990, Macromolecules, 23:3399; all of which areincorporated herein by reference). Examples of these polyesters includepoly(L-lactide-co-L-lysine) (Barrera et al., 1993, J. Am. Chem. Soc.,115:11010; incorporated herein by reference), poly(serine ester) (Zhouet al., 1990, Macromolecules, 23:3399; incorporated herein byreference), poly(4-hydroxy-L-proline ester) (Putnam et al., 1999,Macromolecules, 32:3658 ; and Lim et al., 1999, J. Am. Chem. Soc.,121:5633; both of which are incorporated herein by reference), andpoly(4-hydroxy-L-proline ester) (Putnam et al., 1999, Macromolecules,32:3658; and Lim et al., 1999, J. Am. Chem. Soc., 121:5633; both ofwhich are incorporated herein by reference).

In some embodiments, polymers in accordance with the present inventionmay be carbohydrates, properties of which are described in furtherdetail below. In some embodiments, a carbohydrate may be apolysaccharide comprising simple sugars (or their derivatives) connectedby glycosidic bonds, as known in the art. In some embodiments, acarbohydrate may be one or more of pullulan, cellulose, microcrystallinecellulose, hydroxypropyl methylcellulose, hydroxycellulose,methylcellulose, dextran, cyclodextran, glycogen, starch,hydroxyethylstarch, carageenan, glycon, amylose, chitosan,N,O-carboxylmethylchitosan, algin and alginic acid, starch, chitin,heparin, konjac, glucommannan, pustulan, heparin, hyaluronic acid,curdlan, and xanthan.

In some embodiments, a polymer in accordance with the present inventionmay be a protein or peptide, properties of which are described infurther detail below. Exemplary proteins that may be used in accordancewith the present invention include, but are not limited to, albumin,collagen, a poly(amino acid) (e.g., polylysine), an antibody, etc.

In some embodiments, a polymer in accordance with the present inventionmay be a nucleic acid (i.e., polynucleotide), properties of which aredescribed in further detail below. Exemplary polynucleotides that may beused in accordance with the present invention include, but are notlimited to, DNA, RNA, etc.

In some embodiments, the present invention relates to the use ofpolymeric nanoparticle-nicotine bioconjugate systems as a platform toinduce anti-nicotine antibodies. A controlled release polymer system orvesicle based system, as used herein, is a polymer combined with anactive agent, such as a therapeutic agent, a diagnostic agent, aprognostic, or prophylactic agent, so that the active agent is releasedand/or triggered from the material in a predesigned manner. Thepolymer-nicotine bioconjugates system may be synthesized as ahomopolymer, diblock triblock and/or multiblock copolymer. The synthesisof the polymer-nicotine bioconjugates system includes polymerizationfrom monomers and conjugation of different polymers. The nanoparticlesystem may include a polymer that is biologically degradable, chemicallydegradable, or both biologically and chemically degradable. Examples ofsuitable polymers for controlled release polymer systems include, butare not limited to, poly(lactic acid), derivatives of poly(lactic acid),PEGylated poly(lactic acid), polylactic-co-glycolic acid), derivativesof poly(lacticco-glycolic acid), PEGylated polylactic-co-glycolic acid),poly(anhydrides), PEGylated poly(anhydrides), poly(ortho esters)derivatives of poly(ortho esters), PEGylated poly(ortho esters),poly(caprolactones), derivatives of poly(caprolactone), PEGylatedpoly(caprolactones), polylysine, derivatives of polylysine, PEGylatedpolylysine, poly(ethylene imine), derivatives of poly(ethylene imine),PEGylated poly(ethylene imine), poly(acrylic acid), derivatives ofpoly(acrylic acid), PEGylated poly(acrylic acid), poly(urethane),PEGylated poly(urethane), derivatives of poly(urethane), andcombinations thereof.

The properties of these and other polymers and methods for preparingthem are well known in the art (see, for example, U.S. Pat. Nos.6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404; 6,095,148;5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600; 5,399,665;5,019,379; 5,010,167; 4,806,621; 4,638,045; and 4,946,929; Wang et al.,2001, J. Am. Chem. Soc., 123:9480; Lim et al., 2001, J. Am. Chem. Soc.,123:2460; Langer, 2000, Acc. Chem. Res., 33:94; Langer, 1999, J.Control. Release, 62:7; and Uhrich et al., 1999, Chem. Rev., 99:3181;all of which are incorporated herein by reference). More generally, avariety of methods for synthesizing suitable polymers are described inConcise Encyclopedia of Polymer Science and Polymeric Amines andAmmonium Salts, Ed. by Goethals, Pergamon Press, 1980; Principles ofPolymerization by Odian, John Wiley & Sons, Fourth Edition, 2004;Contemporary Polymer Chemistry by Allcock et al., Prentice-Hall, 1981;Deming et al., 1997, Nature, 390:386; and in U.S. Pat. Nos. 6,506,577,6,632,922, 6,686,446, and 6,818,732; all of which are incorporatedherein by reference.

In some embodiments, polymers can be linear or branched polymers. Insome embodiments, polymers can be dendrimers. In some embodiments,polymers can be substantially cross-linked to one another. In someembodiments, polymers can be substantially free of cross-links. In someembodiments, polymers can be used in accordance with the presentinvention without undergoing a cross-linking step.

It is further to be understood that inventive nanocarriers may compriseblock copolymers, graft copolymers, blends, mixtures, and/or adducts ofany of the foregoing and other polymers.

Those skilled in the art will recognize that the polymers listed hereinrepresent an exemplary, not comprehensive, list of polymers that can beof use in accordance with the present invention.

In some embodiments, vaccine nanocarriers comprise immunomodulatoryagents embedded within reverse micelles. To give but one example, aliposome nanocarrier may comprise hydrophobic immunomodulatory agentsembedded within the liposome membrane, and hydrophilic immunomodulatoryagents embedded with reverse micelles found in the interior of theliposomal nanocarrier.

Non-Polymeric Nanocarriers

In some embodiments, nanocarriers may not comprise a polymericcomponent. In some embodiments, nanocarriers may comprise metalparticles, quantum dots, ceramic particles, bone particles, viralparticles, etc. In some embodiments, an immunomodulatory agent,targeting moiety, and/or immunostimulatory agent can be associated withthe surface of such a non-polymeric nanocarrier. In some embodiments, anon-polymeric nanocarrier is an aggregate of non-polymeric components,such as an aggregate of metal atoms (e.g., gold atoms). In someembodiments, an immunomodulatory agent, targeting moiety, and/orimmunostimulatory agent can be associated with the surface of,encapsulated within, surrounded by, and/or dispersed throughout anaggregate of non-polymeric components.

In certain embodiments of the invention, non-polymeric nanocarrierscomprise gradient or homogeneous alloys. In certain embodiments of theinvention, nanocarriers comprise particles which possess opticallyand/or magnetically detectable properties.

Nanocarriers Comprising Amphiphilic Entities

In some embodiments, nanocarriers may optionally comprise one or moreamphiphilic entities. In some embodiments, an amphiphilic entity canpromote the production of nanocarriers with increased stability,improved uniformity, or increased viscosity. In some embodiments,amphiphilic entities can be associated with the interior surface of alipid membrane (e.g., lipid bilayer, lipid monolayer, etc.). Forexample, if the interior surface of a lipid membrane is hydrophilic, thespace encapsulated within the lipid nanocarrier is hydrophilic. However,if an amphiphilic entity is associated with the interior surface of thehydrophilic lipid membrane such that the hydrophilic end of theamphiphilic entity is associated with the interior surface of thehydrophilic lipid membrane and the hydrophobic end of the amphiphilicentity is associated with the interior of the nanocarrier, the spaceencapsulated within the nanocarrier is hydrophobic.

The percent of amphiphilic entity in nanocarriers can range from 0% to99% by weight, from 10% to 99% by weight, from 25% to 99% by weight,from 50% to 99% by weight, or from 75% to 99% by weight. In someembodiments, the percent of amphiphilic entity in nanocarriers can rangefrom 0% to 75% by weight, from 0% to 50% by weight, from 0% to 25% byweight, or from 0% to 10% by weight. In some embodiments, the percent ofamphiphilic entity in nanocarriers can be approximately 1% by weight,approximately 2% by weight, approximately 3% by weight, approximately 4%by weight, approximately 5% by weight, approximately 10% by weight,approximately 15% by weight, approximately 20% by weight, approximately25% by weight, or approximately 30% by weight.

Any amphiphilic entity known in the art is suitable for use in makingnanocarriers in accordance with the present invention. Such amphiphilicentities include, but are not limited to, phosphoglycerides;phosphatidylcholines; dipalmitoyl phosphatidylcholine (DPPC);dioleylphosphatidyl ethanolamine (DOPE);dioleyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine;cholesterol; cholesterol ester; diacylglycerol; diacylglycerolsuccinate;diphosphatidyl glycerol (DPPG); hexanedecanol; fatty alcohols such aspolyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; a surfaceactive fatty acid, such as palmitic acid or oleic acid; fatty acids;fatty acid monoglycerides; fatty acid diglycerides; fatty acid amides;sorbitan trioleate (Span® 85) glycocholate; sorbitan monolaurate (Span®20); polysorbate 20 (Tween® 20); polysorbate 60 (Tween® 60); polysorbate65 (Tween® 65); polysorbate 80 (Tween® 80); polysorbate 85 (Tween® 85);polyoxyethylene monostearate; surfactin; a poloxomer; a sorbitan fattyacid ester such as sorbitan trioleate; lecithin; lysolecithin;phosphatidylserine; phosphatidylinositol; sphingomyelin;phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic acid;cerebrosides; dicetylphosphate; dipalmitoylphosphatidylglycerol;stearylamine; dodecylamine; hexadecyl-amine; acetyl palmitate; glycerolricinoleate; hexadecyl stearate; isopropyl myristate; tyloxapol;poly(ethylene glycol)5000-phosphatidylethanolamine; poly(ethyleneglycol)400-monostearate; phospholipids; synthetic and/or naturaldetergents having high surfactant properties; deoxycholates;cyclodextrins; chaotropic salts; ion pairing agents; and combinationsthereof. An amphiphilic entity component may be a mixture of differentamphiphilic entities. These amphiphilic entities may be extracted andpurified from a natural source or may be prepared synthetically in alaboratory. In certain specific embodiments, amphiphilic entities arecommercially available.

Those skilled in the art will recognize that this is an exemplary, notcomprehensive, list of substances with surfactant activity. Anyamphiphilic entity may be used in the production of nanocarriers to beused in accordance with the present invention.

Vaccine Nanocarriers Comprising Carbohydrates

In some embodiments, nanocarriers may optionally comprise one or morecarbohydrates. The percent of carbohydrate in nanocarriers can rangefrom 0% to 99% by weight, from 10% to 99% by weight, from 25% to 99% byweight, from 50% to 99% by weight, or from 75% to 99% by weight. In someembodiments, the percent of carbohydrate in nanocarriers can range from0% to 75% by weight, from 0% to 50% by weight, from 0% to 25% by weight,or from 0% to 10% by weight. In some embodiments, the percent ofcarbohydrate in nanocarriers can be approximately 1% by weight,approximately 2% by weight, approximately 3% by weight, approximately 4%by weight, approximately 5% by weight, approximately 10% by weight,approximately 15% by weight, approximately 20% by weight, approximately25% by weight, or approximately 30% by weight.

Carbohydrates may be natural or synthetic. A carbohydrate may be aderivatized natural carbohydrate. In certain embodiments, a carbohydrateis a monosaccharide, including but not limited to glucose, fructose,galactose, ribose, lactose, sucrose, maltose, trehalose, cellbiose,mannose, xylose, arabinose, glucoronic acid, galactoronic acid,mannuronic acid, glucosamine, galatosamine, and neuramic acid. Incertain embodiments, a carbohydrate is a disaccharide, including but notlimited to lactose, sucrose, maltose, trehalose, and cellobiose. Incertain embodiments, a carbohydrate is a polysaccharide, including butnot limited to pullulan, cellulose, microcrystalline cellulose,hydroxypropyl methylcellulose (HPMC), hydroxycellulose (HC),methylcellulose (MC), dextran, cyclodextran, glycogen, starch,hydroxyethylstarch, carageenan, glycon, amylose, chitosan,N,O-carboxylmethylchitosan, algin and alginic acid, starch, chitin,heparin, konjac, glucommannan, pustulan, heparin, hyaluronic acid,curdlan, and xanthan. In certain embodiments, the carbohydrate is asugar alcohol, including but not limited to mannitol, sorbitol, xylitol,erythritol, maltitol, and lactitol.

Particles and Particles Associated with Vaccine Nanocarriers

In some embodiments, vaccine nanocarriers in accordance with the presentinvention may comprise one or more particles. In some embodiments, oneor more particles are associated with a vaccine nanocarrier. In someembodiments, vaccine nanocarriers comprise one or more particlesassociated with the outside surface of the nanocarrier. In someembodiments, particles may be associated with vaccine nanocarriers viacovalent linkage. In some embodiments, particles may be associated withvaccine nanocarriers via non-covalent interactions (e.g., chargeinteractions, affinity interactions, metal coordination, physicaladsorption, host-guest interactions, hydrophobic interactions, TTstacking interactions, hydrogen bonding interactions, van der Waalsinteractions, magnetic interactions, electrostatic interactions,dipole-dipole interactions, and/or combinations thereof). In someembodiments, vaccine nanocarriers comprise one or more particlesencapsulated within the nanocarrier. In some embodiments, vaccinenanocarriers comprise one or more particles embedded within the surfaceof the nanocarrier (e.g., embedded within a lipid bilayer). In someembodiments, particles associated with a nanocarrier allow for tunablemembrane rigidity and controllable liposome stability.

In some embodiments, vaccine nanocarrier particles or particles to beassociated with a vaccine nanocarrier may comprise a polymeric matrix,as described above. In some embodiments, vaccine nanocarrier particlesor particles to be associated with a vaccine nanocarrier may comprisenon-polymeric components (e.g., metal particles, quantum dots, ceramicparticles, bone particles, viral particles, etc.), as described above.

In some embodiments, vaccine nanocarrier particles or particles to beassociated with a vaccine nanocarrier may have a negative charge. Insome embodiments, vaccine nanocarrier particles or particles to beassociated with a vaccine nanocarrier may have a positive charge. Insome embodiments, vaccine nanocarrier particles or particles to beassociated with a vaccine nanocarrier may be electrically neutral.

In some embodiments, the particles have one or more amine moieties on asurface. The amine moieties can be, for example, aliphatic aminemoieties. In certain embodiments, the amine is a primary, secondary,tertiary, or quaternary amine. In certain embodiments, the particlecomprises an amine-containing polymer. In certain embodiments, theparticle comprises an amine-containing lipid. In certain embodiments,the particles comprises a protein or a peptide that is positivelycharged at neutral pH. In some embodiments, the particle with the one ormore amine moieties on its surface has a net positive charge at neutralpH. Other chemical moieties that provide a positive charge at neutral pHmay also be used in the inventive particles.

In some embodiments, the particles have one or more negatively charged(at neutral pH) moieties on a surface. For example, the particles haveone or more carboxylic acid or phosphoric acid groups on the surface. Insome embodiments, the particle with the one or more carboxylic acid orphosphoric acid moieties on its surface has a net negative charge atneutral pH. Other chemical moieties that provide a negative charge atneutral pH may also be used in the inventive particles.

In some embodiments, the particles have one or more substantiallyneutral (at neutral pH) moieties on a surface. For example, theparticles have one or more ether groups on the surface. In someembodiments, the particle with the one or more ether moieties on itssurface has substantially no net charge at neutral pH. Other chemicalmoieties that provide a neutral charge at neutral pH may also be used inthe inventive particles.

Some non-limiting examples of compounds that can be present at thesurface of the nanocarriers in order to effect the charge at the surfaceinclude 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-dioleoyl-3-trimethylammonium-propane, chloride salt (DOTAP),monosialoganglioside GM3,1,2-dihexadecanoyl-sn-glycero-3-phospho-L-serine, sodium salt (DPPS),monophosphoryl Lipid A, and N-4-nitrobenzo-2-oxa-1,3-diazolephosphatidylethanolamine (NBD-PE).

Zeta potential is a measurement of surface potential of a particle. Insome embodiments, the nanocarrier particles or particles associated withthe nanocarriers have a positive zeta potential. In some embodiments,particles have a zeta potential ranging between −50 mV and +50 mV. Insome embodiments, particles have a zeta potential ranging between −25 mVand +25 mV. In some embodiments, particles have a zeta potential rangingbetween −10 mV and +10 mV. In some embodiments, particles have a zetapotential ranging between −5 mV and +5 mV. In some embodiments,particles have a zeta potential ranging between 0 mV and +50 mV. In someembodiments, particles have a zeta potential ranging between 0 mV and+25 mV. In some embodiments, particles have a zeta potential rangingbetween 0 mV and +10 mV. In some embodiments, particles have a zetapotential ranging between 0 mV and +5 mV. In some embodiments, particleshave a zeta potential ranging between −50 mV and 0 mV. In someembodiments, particles have a zeta potential ranging between −25 mV and0 mV. In some embodiments, particles have a zeta potential rangingbetween −10 mV and 0 mV. In some embodiments, particles have a zetapotential ranging between −5 mV and 0 mV. In some embodiments, particleshave a substantially neutral zeta potential (i.e. approximately 0 mV).

Particles (e.g., nanoparticles, microparticles) may be prepared usingany method known in the art. For example, particulate formulations canbe formed by methods as nanoprecipitation, flow focusing fluidicchannels, spray drying, single and double emulsion solvent evaporation,solvent extraction, phase separation, milling, microemulsion procedures,microfabrication, nanofabrication, sacrificial layers, simple andcomplex coacervation, and other methods well known to those of ordinaryskill in the art. Alternatively or additionally, aqueous and organicsolvent syntheses for monodisperse semiconductor, conductive, magnetic,organic, and other nanoparticles have been described (Pellegrino et al.,2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci., 30:545; andTrindade et al., 2001, Chem. Mat., 13:3843; all of which areincorporated herein by reference).

In certain embodiments, particles are prepared by the nanoprecipitationprocess or spray drying. Conditions used in preparing particles may bealtered to yield particles of a desired size or property (e.g.,hydrophobicity, hydrophilicity, external morphology, “stickiness,”shape, etc.). The method of preparing the particle and the conditions(e.g., solvent, temperature, concentration, air flow rate, etc.) usedmay depend on the therapeutic agent to be delivered and/or thecomposition of the polymer matrix.

Methods for making microparticles for delivery of encapsulated agentsare described in the literature (see, e.g., Doubrow, Ed., “Microcapsulesand Nanoparticles in Medicine and Pharmacy,” CRC Press, Boca Raton,1992; Mathiowitz et al., 1987, J. Control. Release, 5:13; Mathiowitz etal., 1987, Reactive Polymers, 6: 275; and Mathiowitz et al., 1988, J.Appl. Polymer Sci., 35:755; all of which are incorporated herein byreference).

If particles prepared by any of the above methods have a size rangeoutside of the desired range, particles can be sized, for example, usinga sieve.

Immunofeature Surface

The synthetic nanocarriers of the invention comprise one or moresurfaces, and at least one surface comprises an immunofeature surface.An immunofeature surface is a surface that comprises multiple moieties,wherein: (1) the immunofeature surface excludes moieties that are the Fcportion of an antibody; and (2) the moieties are present in an amounteffective to provide binding to either human dendritic cells or murinesubcapsular sinus macrophages (collectively “In Vitro Antigen PresentingCells”) such that when subjected to an in vitro assay that compares theimmobilization of the In Vitro Antigen Presenting Cells on abiocompatible surface that is coated with either the moieties of whichthe immunofeature surface is comprised, or an antibody that is specificfor an In Vitro Antigen Presenting Cell-expressed surface antigen (forhuman dendritic cells: anti-CD1c (BDCA-1) clone AD5-8E7 from MiltenyiBiotec®, or for murine subcapsular sinus macrophages: anti-CD169 clone3D6.112 from Serotec®), (i) an optimal coating density corresponding tomaximal immobilization of the In Vitro Antigen Presenting Cells to thesurface which has been coated with the moieties of which theimmunofeature surface is comprised is at least 10%, preferably at least20%, more preferably at least 25%, of that observed with the antibodycoated surface; and (ii) the immunofeature surface that is being testedsupports half maximal binding at a coating density of moieties of whichthe immunofeature surface is comprised that is at least 2-fold,preferably at least 3-fold, more preferably at least 4-fold higher thanthe antibody coating density that supports half maximal binding (thistype of binding may also be referred to as “high avidity” binding).

Immunofeature surfaces may be positively charged, negatively charged orneutrally charged at pH=7.2-7.4. Immunofeature surfaces may be made upof the same moiety or a mixture of different moieties. In embodiments,the immunofeature surfaces may comprise B cell antigens. Examples ofmoieties potentially useful in immunofeature surfaces comprise: methoxygroups, positively charged amine groups (e.g. tertiary amines),sialyllactose, avidin and/or avidin derivatives such as NeutrAvidin, andresidues of any of the above. In an embodiment, the moieties of whichthe immunofeature surface is comprised are coupled to a surface of theinventive nanocarriers. In another embodiment, the immunofeature surfaceis coupled to a surface of the inventive nanocarriers.

It should be noted that moieties of which immunofeature surfaces arecomprised confer high avidity binding. Not all moieties that could bepresent on a nanocarrier will confer high avidity binding, as definedspecifically in this definition, and described generally throughout thepresent specification. Accordingly, even though a surface may comprisemultiple moieties (sometimes referred to as an “array”), this does notmean that such a surface inherently is an immunofeature surface absentdata showing that such a surface confers binding according to thepresent definition and disclosure.

An in vitro test for the assessment of avidity of immunofeature surfacesthat target immunonanotherapeutics to antigen-presenting cells in lymphnodes may be performed by comparing the immobilization of In VitroAntigen Presenting Cells on biocompatible surfaces that are coated witheither the moieties of which the immunofeature surface is comprised oran antibody that is specific for a In Vitro Antigen PresentingCells-expressed surface antigen. In an aspect of the invention, theassay is as follows:

1. Both the antibodies (anti-CD1c (BDCA-1) clone AD5-8E7 from MiltenyiBiotec®, a MAb that recognizes and is used to assay for human dendriticcells, or anti-CD169 clone 3D6.112 from Serotec®, a MAb that recognizesand is used to assay for murine subcapsular sinus macrophages) and themoieties of which the immunofeature surface is comprised are immobilizedat a range of coating densities (e.g. 100/mm² to 1×10E12/mm²) on asuitable substrate, such as a glass slide or a plastic dish used formammalian tissue culture, that supports little or no spontaneousadhesion of In Vitro Antigen Presenting Cells.

2. Single-cell suspensions of purified human dendritic cells or murinesubcapsular sinus, preferably CD169-high, macrophages (1×10E6/ml) areadded to the coated surfaces at 37° C. under gentle agitation on anorbital shaker. After 15-30 minutes, nonadherent cells are removed bygentle washing (e.g. dipping into a jar containing ice-cold HBSS medium)and the plates are fixed with ice-cold glutaraldehyde.

3. The number of immobilized In Vitro Antigen Presenting Cells per unitsurface area are counted by phase contrast microscopy or similarquantitative techniques.

4. The number of immobilized cells/field is then plotted against thecoating density of the antibody used and the coating density of themoieties of which the immunofeature surface is comprised.

5. Optimal coating densities that correspond to a maximal immobilizationof the In Vitro Antigen Presenting Cells by the coated antibody and bythe coated moieties of which the immunofeature surface is comprised aredetermined.

6. The coating densities that support 50% of maximal immobilization(“half-maximal binding”) of the In Vitro Antigen Presenting Cells by thecoated antibody and the coated moieties of which the immunofeaturesurface is comprised are determined.

In some embodiments, the plurality of moieties are other than the Fcportion of an antibody. In some embodiments, the plurality of moietiesare selected from immunostimulatory moieties (as described herein),immunomodulatory moieties (as described herein), targeting moieties (asdescribed herein), small organic moieties (e.g., nicotine), oligomers,and polymers (including synthetic polymers such as PEG), inorganicmoieties, nucleic acids and polynucleotides (e.g., DNA and RNAfragments), amino acids, polypeptides, glycoproteins, biologicallyactive substances (as described herein), gangliosides, lipids,phospholipids, carbohydrates, polysaccharides, and fragments of any ofthe foregoing. In some embodiments, the immunofeature surfaces maycomprise B-cell antigens or T-cell antigens. Any combination of theforegoing moieties are also within the scope of the invention. It willbe appreciated that bonding of such moieties to the immunofeaturesurface may involve appropriate modification such as replacement of abond to an atom in the moiety with a bond to the surface.

In some embodiments, bonding of the plurality of moieties to theimmunofeature surface is via covalent bonds between the moiety and acomponent of the nanocarrier. For example, in the case of nanocarrierscomprising polymeric materials, the plurality of moieties may becovalently bonded to the polymers. In some cases, however, non-covalentinteractions may be used, including ionic or hydrogen bonding, ordispersion forces.

Immunofeature surfaces may comprise a plurality of moieties that are thesame moiety or a mixture of different moieties. For example, 2, 3, 4, 5,6, 7, 8, 9, or 10 different types of moieties may be present in theimmunofeature surface.

Immunofeature surfaces may be overall positively charged, negativelycharged or neutrally charged when the immunofeature surface is presentin buffered aqueous solution at pH in the range 7.2-7.4. It will beappreciated that, where a mixture of different moieties are present onethe immunofeature surface, the immunofeature surface may be associatedwith a combination of positively charged, negatively charged, and/orneutrally charged moieties at any particular pH.

The immunofeature surface provides high avidity binding to an antigenpresenting cell (APC), particularly APCs expressing MHC II molecules.For example, in some embodiments, the immunofeature surface provideshigh avidity binding to dendritic cells (DCs). In some otherembodiments, the immunofeature surface provides high avidity binding tomacrophages, for example subcapsular sinus macrophages (SCS Mphs). Insome embodiments, the immunofeature surface provides high aviditybinding to B-cells (i.e., the immunofeature surfaces comprises B-cellantigens). In some embodiments the immunofeature surface provides highavidity and low affinity binding to DCs, SCS Mphs, or B-cells.

Immunofeature surfaces comprise a plurality of moieties, and theplurality of moieties may be all the same moiety or a mixture ofdifferent moieties. The plurality of moietiesare present on a surfacethat is capable of binding to APC surfaces with high avidity. In someembodiments, The plurality of moietiesare present on a surface that iscapable of binding to APC surfaces with high avidity and low affinity.Examples of moieties useful in immunofeature surfaces include: smallorganic molecules such as nicotine, mannose, maleimide, POPC, DPPS,DOPS, monophosphoryl Lipid A, NBD-PE, and derivatives thereof;functional groups such as methoxy, amine, carboxylic acid, and analoguesthereof; polysaccharides such as silalylactose and monosialogangliosideGM3; proteins and polypeptides such as avidin, NeutrAvidin, lysozyme,oligomer G, protein G, and derivatives thereof, and the like.

The extent to which the moieties present in an immunofeature surface arecapable of directing nanocarriers to APCs will vary. For example,moieties that provide an immunofeature surface that is more specificallytargeted to APCs (i.e., more specifically able to bind to APCs with highavidity and low affinity) will result in nanocarrier formulations thatexhibit greater accumulation in areas such as the SCS.

It should be noted that moieties of which immunofeature surfaces arecomprised confer high avidity binding. Not all moieties that are presenton a nanocarrier will confer high avidity binding, as set forthspecifically in this definition, and generally throughout the presentspecification. Accordingly, even though a surface may comprise multiplemoieties (sometimes referred to as an “array”), this does not mean thatsuch a surface inherently is an immunofeature surface absent datashowing that such a surface confers binding according to the presentdefinition and disclosure.

In some embodiments, nanocarriers having a surface comprising animmunofeature surface are able to target specific APCs when administeredto a subject. For example, in some embodiments, the nanocarriers with animmunofeature surface are able to target DCs. In some embodiments, thenanocarriers with an immunofeature surface are able to targetmacrophages, such as SCS-Mphs. In some embodiments, the nanocarrierswith an immunofeature surface are able to target B-cells. In thiscontext, the terms “target” are used to indicate that compositions ofnanocarriers having an immunofeature surface tend to accumulate in aspecific region upon administration to a subject. Such accumulation mayoccur within minutes (e.g., less than 1 minute, less than 5 minutes, orless than 10 minutes, or less than 30 minutes), hours (e.g., less than 1hour, or less than 2 hours, or less than 5 hours, or less than 10hours), or days (e.g., less than 1 day, or less than 2 days, or lessthan 5 days) of administration.

In some embodiments, the plurality of moieties on the immunofeaturesurface includes nicotine. In such embodiments, the nicotine moitiesserve at least two functions: (1) the nicotine moieties providetargeting of the nanocarriers to SCS-Mphs; and (2) the nicotine moietiesserve as B-cell antigens to stimulate the production of anti-nicotineantibodies.

Production of Nanocarriers

Synthetic nanocarriers may be prepared using any method known in theart. For example, particulate nanocarrier formulations can be formed bymethods as nanoprecipitation, flow focusing fluidic channels, spraydrying, single and double emulsion solvent evaporation, solventextraction, phase separation, milling, microemulsion procedures,microfabrication, nanofabrication, sacrificial layers, simple andcomplex coacervation, and other methods well known to those of ordinaryskill in the art. Alternatively or additionally, aqueous and organicsolvent syntheses for monodisperse semiconductor, conductive, magnetic,organic, and other nanoparticles have been described (Pellegrino et al.,2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci., 30:545; andTrindade et al., 2001, Chem. Mat., 13:3843; all of which areincorporated herein by reference).

In certain embodiments, synthetic nanocarriers are prepared by thenanoprecipitation process or spray drying. Conditions used in preparingnanocarriers may be altered to yield particles of a desired size orproperty (e.g., hydrophobicity, hydrophilicity, external morphology,“stickiness,” shape, etc.). The method of preparing the nanocarrier andthe conditions (e.g., solvent, temperature, concentration, air flowrate, etc.) used may depend on the composition and/or resultingarchitecture of the nanocarrier.

Methods useful in making synthetic nanocarriers according to theinvention are described in the literature (see, e.g., Doubrow, Ed.,“Microcapsules and Nanoparticles in Medicine and Pharmacy,” CRC Press,Boca Raton, 1992; Mathiowitz et al., 1987, J. Control. Release, 5:13;Mathiowitz et al., 1987, Reactive Polymers, 6: 275; and Mathiowitz etal., 1988, J. Appl. Polymer Sci., 35:755; all of which are incorporatedherein by reference).

In some embodiments, inventive vaccine nanocarriers comprise at leastone immunomodulatory agent and, optionally, a lipid membrane, apolymeric matrix, and/or a non-polymeric particle. In certainembodiments, inventive vaccine nanocarriers comprise at least oneimmunomodulatory agent; a lipid membrane, a polymeric matrix, and/or anon-polymeric particle; and at least one targeting moiety. In certainembodiments, inventive vaccine nanocarriers comprise at least oneimmunomodulatory agent; a lipid membrane, a polymeric matrix, and/or anon-polymeric particle; at least one targeting moiety; and at least oneimmunostimulatory agent. In certain embodiments, inventive vaccinenanocarriers comprise at least one immunomodulatory agent; a lipidmembrane, a polymeric matrix, and/or a non-polymeric particle; at leastone targeting moiety; at least one immunostimulatory agent; and at leastone nanoparticle.

Inventive nanocarriers may be manufactured using any available method.It is desirable to associate immunomodulatory agents, targetingmoieties, and/or immunostimulatory agents to vaccine nanocarrierswithout adversely affecting the 3-dimensional characteristic andconformation of the immunomodulatory agents, targeting moieties, and/orimmunostimulatory agents. It is desirable that the vaccine nanocarriershould be able to avoid uptake by the mononuclear phagocytic systemafter systemic administration so that it is able to reach specific cellsin the body.

In some embodiments, immunomodulatory agents, targeting moieties,immunostimulatory agents, and/or nanoparticles are not covalentlyassociated with a vaccine nanocarrier. For example, vaccine nanocarriersmay comprise a polymeric matrix, and immunomodulatory agents, targetingmoieties, immunostimulatory agents, and/or nanoparticles may beassociated with the surface of, encapsulated within, and/or distributedthroughout the polymeric matrix of an inventive vaccine nanocarrier.Immunomodulatory agents are released by diffusion, degradation of thevaccine nanocarrier, and/or combination thereof. In some embodiments,polymers degrade by bulk erosion. In some embodiments, polymers degradeby surface erosion.

In some embodiments, immunomodulatory agents, targeting moieties,immunostimulatory agents, and/or nanoparticles are covalently associatedwith a vaccine nanocarrier. For such vaccine nanocarriers, release anddelivery of the immunomodulatory agent to a target site occurs bydisrupting the association. For example, if an immunomodulatory agent isassociated with a nanocarrier by a cleavable linker, theimmunomodulatory agent is released and delivered to the target site uponcleavage of the linker.

In some embodiments, immunomodulatory agents, targeting moieties,immunostimulatory agents, and/or nanoparticles are not covalentlyassociated with a vaccine nanocarrier. For example, vaccine nanocarriersmay comprise polymers, and immunomodulatory agents, targeting moieties,immunostimulatory agents, and/or nanoparticles may be associated withthe surface of, encapsulated within, surrounded by, and/or distributedthroughout the polymer of an inventive vaccine nanocarrier. In someembodiments, immunomodulatory agents, targeting moieties,immunostimulatory agents, and/or nanoparticles are physically associatedwith a vaccine nanocarrier.

Physical association can be achieved in a variety of different ways.Physical association may be covalent or non-covalent. The vaccinenanocarrier, immunomodulatory agent, targeting moiety, immunostimulatoryagent, and/or nanoparticle may be directly associated with one another,e.g., by one or more covalent bonds, or may be associated by means ofone or more linkers. In one embodiment, a linker forms one or morecovalent or non-covalent bonds with the immunomodulatory agent,targeting moiety, immunostimulatory agent, and/or nanoparticle and oneor more covalent or non-covalent bonds with the immunomodulatory agent,targeting moiety, immunostimulatory agent, and/or nanoparticle, therebyattaching them to one another. In some embodiments, a first linker formsa covalent or non-covalent bond with the vaccine nanocarrier and asecond linker forms a covalent or non-covalent bond with theimmunomodulatory agent, targeting moiety, immunostimulatory agent,and/or nanoparticle. The two linkers form one or more covalent ornon-covalent bond(s) with each other.

Any suitable linker can be used in accordance with the presentinvention. Linkers may be used to form amide linkages, ester linkages,disulfide linkages, etc. Linkers may contain carbon atoms or heteroatoms(e.g., nitrogen, oxygen, sulfur, etc.). Typically, linkers are 1 to 50atoms long, 1 to 40 atoms long, 1 to 25 atoms long, 1 to 20 atoms long,1 to 15 atoms long, 1 to 10 atoms long, or 1 to 10 atoms long. Linkersmay be substituted with various substituents including, but not limitedto, hydrogen atoms, alkyl, alkenyl, alkynyl, amino, alkylamino,dialkylamino, trialkylamino, hydroxyl, alkoxy, halogen, aryl,heterocyclic, aromatic heterocyclic, cyano, amide, carbamoyl, carboxylicacid, ester, thioether, alkylthioether, thiol, and ureido groups. Aswould be appreciated by one of skill in this art, each of these groupsmay in turn be substituted.

In some embodiments, a linker is an aliphatic or heteroaliphatic linker.In some embodiments, the linker is a polyalkyl linker. In certainembodiments, the linker is a polyether linker. In certain embodiments,the linker is a polyethylene linker. In certain specific embodiments,the linker is a polyethylene glycol (PEG) linker.

In some embodiments, the linker is a cleavable linker. To give but a fewexamples, cleavable linkers include protease cleavable peptide linkers,nuclease sensitive nucleic acid linkers, lipase sensitive lipid linkers,glycosidase sensitive carbohydrate linkers, pH sensitive linkers,hypoxia sensitive linkers, photo-cleavable linkers, heat-labile linkers,enzyme cleavable linkers (e.g. esterase cleavable linker),ultrasound-sensitive linkers, x-ray cleavable linkers, etc. In someembodiments, the linker is not a cleavable linker.

Any of a variety of methods can be used to associate a linker with avaccine nanocarrier. General strategies include passive adsorption(e.g., via electrostatic interactions), multivalent chelation, highaffinity non-covalent binding between members of a specific bindingpair, covalent bond formation, etc. (Gao et al., 2005, Curr. Op.Biotechnol., 16:63; incorporated herein by reference). In someembodiments, click chemistry can be used to associate a linker with aparticle.

A bifunctional cross-linking reagent can be employed. Such reagentscontain two reactive groups, thereby providing a means of covalentlyassociating two target groups. The reactive groups in a chemicalcross-linking reagent typically belong to various classes of functionalgroups such as succinimidyl esters, maleimides, and pyridyldisulfides.Exemplary cross-linking agents include, e.g., carbodiimides,N-hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA), dimethylpimelimidate dihydrochloride (DMP), dimethylsuberimidate (DMS),3,3′-dithiobispropionimidate (DTBP), N-Succinimidyl3-[2-pyridyldithio]-propionamido (SPDP), succimidyl α-methylbutanoate,biotinamidohexanoyl-6-amino-hexanoic acid N-hydroxy-succinimide ester(SMCC),succinimidyl-[(N-maleimidopropionamido)-dodecaethyleneglycol]ester(NHS-PEO12), etc. For example, carbodiimide-mediated amide formation andactive ester maleimide-mediated amine and sulfhydryl coupling are widelyused approaches.

In some embodiments, a vaccine nanocarrier can be formed by coupling anamine group on one molecule to a thiol group on a second molecule,sometimes by a two- or three-Page step reaction sequence. Athiol-containing molecule may be reacted with an amine-containingmolecule using a heterobifunctional cross-linking reagent, e.g., areagent containing both a succinimidyl ester and either a maleimide, apyridyldisulfide, or an iodoacetamide. Amine-carboxylic acid andthiol-carboxylic acid cross-linking, maleimide-sulfhydryl couplingchemistries (e.g., the maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)method), etc., may be used. Polypeptides can conveniently be attached toparticles via amine or thiol groups in lysine or cysteine side chainsrespectively, or by an N-terminal amino group. Nucleic acids such asRNAs can be synthesized with a terminal amino group. A variety ofcoupling reagents (e.g., succinimidyl 3-(2-pyridyldithio)propionate(SPDP) andsulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-SMCC) may be used to associate the various components of vaccinenanocarriers. Vaccine nanocarriers can be prepared with functionalgroups, e.g., amine or carboxyl groups, available at the surface tofacilitate association with a biomolecule.

Non-covalent specific binding interactions can be employed. For example,either a particle or a biomolecule can be functionalized with biotinwith the other being functionalized with streptavidin. These twomoieties specifically bind to each other non-covalently and with a highaffinity, thereby associating the particle and the biomolecule. Otherspecific binding pairs could be similarly used. Alternately,histidine-tagged biomolecules can be associated with particlesconjugated to nickel-nitrolotriaceteic acid (Ni-NTA).

Any biomolecule to be attached to a particle, targeting moiety, and/ortherapeutic agent. The spacer can be, for example, a short peptidechain, e.g., between 1 and 10 amino acids in length, e.g., 1, 2, 3, 4,or 5 amino acids in length, a nucleic acid, an alkyl chain, etc.

For additional general information on association and/or conjugationmethods and cross-linkers, see the journal Bioconjugate Chemistry,published by the American Chemical Society, Columbus Ohio, PO Box 3337,Columbus, Ohio, 43210; “Cross-Linking,” Pierce Chemical TechnicalLibrary, available at the Pierce web site and originally published inthe 1994-95 Pierce Catalog, and references cited therein; Wong S S,Chemistry of Protein Conjugation and Cross-linking, CRC PressPublishers, Boca Raton, 1991; and Hermanson, G. T., BioconjugateTechniques, Academic Press, Inc., San Diego, 1996.

Alternatively or additionally, vaccine nanocarriers can be attached toimmunomodulatory agents, targeting moieties, immunostimulatory agents,and/or nanoparticles directly or indirectly via non-covalentinteractions. Non-covalent interactions include but are not limited tocharge interactions, affinity interactions, metal coordination, physicaladsorption, host-guest interactions, hydrophobic interactions, TTstacking interactions, hydrogen bonding interactions, van der Waalsinteractions, magnetic interactions, electrostatic interactions,dipole-dipole interactions, and/or combinations thereof.

In some embodiments, a vaccine nanocarrier may be associated with animmunomodulatory agent, targeting moiety, immunostimulatory agent,and/or nanoparticle via charge interactions. For example, a vaccinenanocarrier may have a cationic surface or may be reacted with acationic polymer, such as poly(lysine) or poly(ethylene imine), toprovide a cationic surface. The vaccine nanocarrier surface can thenbind via charge interactions with a negatively charged immunomodulatoryagent, targeting moiety, immunostimulatory agent, and/or nanoparticle.One end of the immunomodulatory agent, targeting moiety,immunostimulatory agent, and/or nanoparticle is, typically, attached toa negatively charged polymer (e.g., a poly(carboxylic acid)) or anadditional oligonucleotide sequence that can interact with the cationicpolymer surface without disrupting the function of the immunomodulatoryagent, targeting moiety, immunostimulatory agent, and/or nanoparticle.

In some embodiments, a vaccine nanocarrier may be associated with animmunomodulatory agent, targeting moiety, immunostimulatory agent,and/or nanoparticle via affinity interactions. For example, biotin maybe attached to the surface of the vaccine nanocarrier and streptavidinmay be attached to the immunomodulatory agent, targeting moiety,immunostimulatory agent, and/or nanoparticle; or conversely, biotin maybe attached to the immunomodulatory agent, targeting moiety,immunostimulatory agent, and/or nanoparticle and the streptavidin may beattached to the surface of the vaccine nanocarrier. The biotin group andstreptavidin may be attached to the vaccine nanocarrier or to theimmunomodulatory agent, targeting moiety, immunostimulatory agent,and/or nanoparticle via a linker, such as an alkylene linker or apolyether linker. Biotin and streptavidin bind via affinityinteractions, thereby binding the vaccine nanocarrier to theimmunomodulatory agent, targeting moiety, immunostimulatory agent,and/or nanoparticle.

In some embodiments, a vaccine nanocarrier may be associated with animmunomodulatory agent, targeting moiety, immunostimulatory agent,and/or nanoparticle via metal coordination. For example, a polyhistidinemay be attached to one end of the immunomodulatory agent, targetingmoiety, immunostimulatory agent, and/or nanoparticle, and anitrilotriacetic acid can be attached to the surface of the vaccinenanocarrier. A metal, such as Ni²⁺, will chelate the polyhistidine andthe nitrilotriacetic acid, thereby binding the immunomodulatory agent,targeting moiety, immunostimulatory agent, and/or nanoparticle to thevaccine nanocarrier.

In some embodiments, a vaccine nanocarrier may be associated with animmunomodulatory agent, targeting moiety, immunostimulatory agent,and/or nanoparticle via physical adsorption. For example, a hydrophobictail, such as polymethacrylate or an alkyl group having at least about10 carbons, may be attached to one end of the immunomodulatory agent,targeting moiety, immunostimulatory agent, and/or nanoparticle. Thehydrophobic tail will adsorb onto the surface of a hydrophobic vaccinenanocarrier, thereby binding the immunomodulatory agent, targetingmoiety, immunostimulatory agent, and/or nanoparticle to the vaccinenanocarrier.

In some embodiments, a vaccine nanocarrier may be associated with animmunomodulatory agent, targeting moiety, immunostimulatory agent,and/or nanoparticle via host-guest interactions. For example, amacrocyclic host, such as cucurbituril or cyclodextrin, may be attachedto the surface of the vaccine nanocarrier and a guest group, such as analkyl group, a polyethylene glycol, or a diaminoalkyl group, may beattached to the immunomodulatory agent, targeting moiety,immunostimulatory agent, and/or nanoparticle; or conversely, the hostgroup may be attached to the immunomodulatory agent, targeting moiety,immunostimulatory agent, and/or nanoparticle and the guest group may beattached to the surface of the vaccine nanocarrier. In some embodiments,the host and/or the guest molecule may be attached to theimmunomodulatory agent, targeting moiety, immunostimulatory agent,and/or nanoparticle or the vaccine nanocarrier via a linker, such as analkylene linker or a polyether linker.

In some embodiments, a vaccine nanocarrier may be associated with animmunomodulatory agent, targeting moiety, immunostimulatory agent,and/or nanoparticle via hydrogen bonding interactions. For example, anoligonucleotide having a particular sequence may be attached to thesurface of the vaccine nanocarrier, and an essentially complementarysequence may be attached to one or both ends of the immunomodulatoryagent, targeting moiety, immunostimulatory agent, and/or nanoparticlesuch that it does not disrupt the function of the immunomodulatoryagent, targeting moiety, immunostimulatory agent, and/or nanoparticle.The immunomodulatory agent, targeting moiety, immunostimulatory agent,and/or nanoparticle then binds to the vaccine nanocarrier viacomplementary base pairing with the oligonucleotide attached to thevaccine nanocarrier. Two oligonucleotides are essentially complimentaryif about 80% of the nucleic acid bases on one oligonucleotide formhydrogen bonds via an oligonucleotide base pairing system, such asWatson-Crick base pairing, reverse Watson-Crick base pairing, Hoogstenbase pairing, etc., with a base on the second oligonucleotide.Typically, it is desirable for an oligonucleotide sequence attached tothe vaccine nanocarrier to form at least about 6 complementary basepairs with a complementary oligonucleotide attached to theimmunomodulatory agent, targeting moiety, immunostimulatory agent,and/or nanoparticle.

In some embodiments, vaccine nanocarriers are made by self-assembly. Fora detailed example of self-assembly of vaccine nanocarriers, seeExamples 1 and 2. In certain embodiments, small liposomes (10 nm-1000nm) are manufactured and employed to deliver one or multipleimmunomodulatory agents to cells of the immune system (FIG. 3). Ingeneral, liposomes are artificially-constructed spherical lipidvesicles, whose controllable diameter from tens to thousands of nmsignifies that individual liposomes comprise biocompatible compartmentswith volume from zeptoliters (10⁻²¹ L) to femtoliters (10⁻¹⁵ L) that canbe used to encapsulate and store various cargoes such as proteins,enzymes, DNA and drug molecules. Liposomes may comprise a lipid bilayerwhich has an amphiphilic property: both interior and exterior surfacesof the bilayer are hydrophilic, and the bilayer lumen is hydrophobic.Lipophilic molecules can spontaneously embed themselves into liposomemembrane and retain their hydrophilic domains outside, and hydrophilicmolecules can be chemically conjugated to the outer surface of liposometaking advantage of membrane biofunctionality.

In certain embodiments, lipids are mixed with a lipophilicimmunomodulatory agent, and then formed into thin films on a solidsurface. A hydrophilic immunomodulatory agent is dissolved in an aqueoussolution, which is added to the lipid films to hydrolyze lipids undervortex. Liposomes with lipophilic immunomodulatory agents incorporatedinto the bilayer wall and hydrophilic immunomodulatory agents inside theliposome lumen are spontaneously assembled.

In certain embodiments, a lipid to be used in liposomes can be, but isnot limited to, one or a plurality of the following:phosphatidylcholine, lipid A, cholesterol, dolichol, sphingosine,sphingomyelin, ceramide, glycosylceramide, cerebroside, sulfatide,phytosphingosine, phosphatidyl-ethanolamine, phosphatidylglycerol,phosphatidylinositol, phosphatidylserine, cardiolipin, phosphatidicacid, and lyso-phophatides. In certain embodiments, an immunomodulatoryagent can be conjugated to the surface of a liposome. In someembodiments, the liposome carries an identical or a non-identicalimmunomodulatory agent inside. In some embodiments, the liposome surfacemembrane can be modified with targeting moieties that can selectivelydeliver the immunomodulatory agent(s) to specific antigen expressingcells.

In some embodiments, nanoparticle-stabilized liposomes are used todeliver one or a plurality of immunomodulatory agents to cells of theimmune system (FIG. 4). By allowing small charged nanoparticles (1 nm-30nm) to adsorb on liposome surface, liposome-nanoparticle complexes havenot only the merits of aforementioned bare liposomes (FIG. 3), but alsotunable membrane rigidity and controllable liposome stability. Whensmall charged nanoparticles approach the surface of liposomes carryingeither opposite charge or no net charge, electrostatic or charge-dipoleinteraction between nanoparticles and membrane attracts thenanoparticles to stay on the membrane surface, being partially wrappedby lipid membrane. This induces local membrane bending and globulesurface tension of liposomes, both of which enable tuning of membranerigidity. This aspect is significant for vaccine delivery usingliposomes to mimic viruses whose stiffness depends on the composition ofother biological components within virus membrane. Moreover, adsorbednanoparticles form a charged shell which protects liposomes againstfusion, thereby enhancing liposome stability. In certain embodiments,small nanoparticles are mixed with liposomes under gentle vortex, andthe nanoparticles stick to liposome surface spontaneously. In specificembodiments, small nanoparticles can be, but are not limited to,polymeric nanoparticles, metallic nanoparticles, inorganic or organicnanoparticles, hybrids thereof, and/or combinations thereof.

In some embodiments, liposome-polymer nanocarriers are used to deliverone or a plurality of immunomodulatory agents to cells of the immunesystem (FIG. 5). Instead of keeping the liposome interior hollow,hydrophilic immunomodulatory agents can be encapsulated. FIG. 3 showsliposomes that are loaded with di-block copolymer nanoparticles to formliposome-coated polymeric nanocarriers, which have the merits of bothliposomes and polymeric nanoparticles, while excluding some of theirlimitations. In some embodiments, the liposome shell can be used tocarry lipophilic or conjugate hydrophilic immunomodulatory agents, andthe polymeric core can be used to deliver hydrophobic immunomodulatoryagents.

In certain embodiments, pre-formulated polymeric nanoparticles (40nm-1000 nm) are mixed with small liposomes (20 nm-100 nm) under gentlevortex to induce liposome fusion onto polymeric nanoparticle surface. Inspecific embodiments, di-block copolymer nanoparticles can be, but arenot limited to, one or a plurality of following: poly(D,Llacticacid)-block-poly(ethylene glycol) (PLA-b-PEG), poly(D,Lglycolicacid)-block-poly(ethylene glycol) (PLG-b-PEG),poly(D,Llactic-co-glycolic acid)-block-poly(ethylene glycol)(PLGA-b-PEG), and poly(ε-caprolactone)-block-poly(ethylene glycol)(PCL-b-PEG).

In some embodiments, nanoparticle-stabilized liposome-polymernanocarriers are used to deliver one or a plurality of immunomodulatoryagents (FIG. 6). By adsorbing small nanoparticles (1 nm-30 nm) to theliposome-polymer nanocarrier surface, the nanocarrier has not only themerit of both aforementioned nanoparticle-stabilized liposomes (FIG. 4)and aforementioned liposome-polymer nanoparticles (FIG. 5), but alsotunable membrane rigidity and controllable liposome stability.

In some embodiments, liposome-polymer nanocarriers containing reversemicelles are used to deliver one or a plurality of immunomodulatoryagents (FIG. 7). Since the aforementioned liposome-polymer nanocarriers(FIGS. 5 and 6) are limited to carry hydrophobic immunomodulatory agentswithin polymeric nanoparticles, here small reverse micelles (1 nm-20 nm)are formulated to encapsulate hydrophilic immunomodulatory agents andthen mixed with the di-block copolymers to formulate polymeric core ofliposomes.

In certain embodiments, a hydrophilic immunomodulatory agent to beencapsulated is first incorporated into reverse micelles by mixing withnaturally derived and non-toxic amphiphilic entities in a volatile,water-miscible organic solvent. In certain embodiments, the amphiphilicentity can be, but is not limited to, one or a plurality of thefollowing: phosphatidylcholine, lipid A, cholesterol, dolichol,sphingosine, sphingomyelin, ceramide, glycosylceramide, cerebroside,sulfatide, phytosphingosine, phosphatidylethanolamine,phosphatidylglycerol, phosphatidylinositol, phosphatidylserine,cardiolipin, phosphatidic acid, and lysophophatides. In someembodiments, the volatile, water-miscible organic solvent can be, but isnot limited to: tetrahydrofuran, acetone, acetonitrile, ordimethylformamide. In some embodiments, a biodegradable polymer is addedto this mixture after reverse micelle formation is complete. Theresulting biodegradable polymer-reverse micelle mixture is combined witha polymer-insoluble hydrophilic non-solvent to form nanoparticles by therapid diffusion of the solvent into the non-solvent and evaporation ofthe organic solvent. In certain embodiments, the polymer-insolublehydrophilic non-solvent can be, but is not limited to one or a pluralityof the following: water, ethanol, methanol, and mixtures thereof.Reverse micelle contained polymeric nanoparticles are mixed with lipidmolecules to form the aforementioned liposome-polymer complex structure(FIG. 5).

In some embodiments, nanoparticle-stabilized liposome-polymernanocarriers containing reverse micelles are used to deliver one or aplurality of immunomodulatory agents (FIG. 8). By adsorbing smallnanoparticles (1 nm-30 nm) to a liposome-polymer nanocarrier surface,the nanocarrier has not only the merit of both aforementionednanoparticle-stabilized liposomes (FIG. 4) and aforementioned reversemicelle contained liposome-polymer nanoparticles (FIG. 7), but alsotunable membrane rigidity and controllable liposome stability.

In some embodiments, lipid monolayer stabilized polymeric nanocarriersare used to deliver one or a plurality of immunomodulatory agents (FIG.9). As compared to aforementioned liposome-polymer nanocarrier (FIGS.5-8), this system has the merit of simplicity in terms to both agentsand manufacturing. In some embodiments, a hydrophobic homopolymer canform the polymeric core in contrast to the di-block copolymer used inFIGS. 5-8, which has both hydrophobic and hydrophilic segments.Lipid-stabilized polymeric nanocarriers can be formed within one singlestep instead of formulating polymeric nanoparticle and liposomeseparately followed by fusing them together.

In certain embodiments, a hydrophilic immunomodulatory molecule is firstchemically conjugated to lipid headgroup. The conjugate is mixed with acertain ratio of unconjugated lipid molecules in an aqueous solutioncontaining one or more water-miscible solvents. In certain embodiments,the amphiphilic entity can be, but is not limited to, one or a pluralityof the following: phosphatidylcholine, lipid A, cholesterol, dolichol,sphingosine, sphingomyelin, ceramide, cerebroside, sulfatide,phytosphingosine, phosphatidylethanolamine, glycosylceramide,phosphatidylglycerol, phosphatidylinositol, phosphatidylserine,cardiolipin, phosphatidic acid, and lysophosphatides. In someembodiments, the water miscible solvent can be, but is not limited to:acetone, ethanol, methanol, and isopropyl alcohol. A biodegradablepolymeric material is mixed with the hydrophobic immunomodulatory agentsto be encapsulated in a water miscible or partially water miscibleorganic solvent. In specific embodiments, the biodegradable polymer canbe, but is not limited to one or a plurality of the following:poly(D,L-lactic acid), poly(D,L-glycolic acid), poly(ε-caprolactone), ortheir copolymers at various molar ratios. In some embodiments, the watermiscible organic solvent can be but is not limited to: acetone, ethanol,methanol, or isopropyl alcohol. In some embodiments, the partially watermiscible organic solvent can be, but is not limited to: acetonitrile,tetrahydrofuran, ethyl acetate, isopropyl alcohol, isopropyl acetate, ordimethylformamide. The resulting polymer solution is added to theaqueous solution of conjugated and unconjugated lipid to yieldnanoparticles by the rapid diffusion of the organic solvent into thewater and evaporation of the organic solvent.

In some embodiments, lipid monolayer stabilized polymeric nanoparticlescomprising reverse micelles are used to deliver one or a plurality ofimmunomodulatory agents (FIG. 10). Since the aforementionedlipid-stabilized polymeric nanocarriers (FIG. 9) are limited to carryhydrophobic immunomodulatory agents, here, small reverse micelles (1nm-20 nm) are formulated to encapsulate hydrophilic immunomodulatoryagents and mixed with biodegradable polymers to form polymericnanocarrier core.

It is to be understood that the compositions of the invention can bemade in any suitable manner, and the invention is in no way limited tocompositions that can be produced using the methods described herein.Selection of an appropriate method may require attention to theproperties of the particular moieties being associated.

If desired, various methods may be used to separate vaccine nanocarrierswith an attached immunomodulatory agent, targeting moiety,immunostimulatory agent, and/or nanoparticle from vaccine nanocarriersto which the immunomodulatory agent, targeting moiety, immunostimulatoryagent, and/or nanoparticle has not become attached, or to separatevaccine nanocarriers having different numbers of immunomodulatoryagents, targeting moieties, immunostimulatory agents, and/ornanoparticles attached thereto. For example, size exclusionchromatography, agarose gel electrophoresis, or filtration can be usedto separate populations of vaccine nanocarriers having different numbersof entities attached thereto and/or to separate vaccine nanocarriersfrom other entities. Some methods include size-exclusion oranion-exchange chromatography.

In some embodiments, inventive vaccine nanocarriers are manufacturedunder sterile conditions. This can ensure that resulting vaccines aresterile and non-infectious, thus improving safety when compared to livevaccines. This provides a valuable safety measure, especially whensubjects receiving vaccine have immune defects, are suffering frominfection, and/or are susceptible to infection.

In some embodiments, inventive vaccine nanocarriers may be lyophilizedand stored in suspension or as lyophilized powder depending on theformulation strategy for extended periods without losing activity.

In some embodiments, the nanocarriers of the invention comprises polymermolecules, and the plurality of moieties of the immunofeature surfaceare associated with the polymer molecules. In preferred embodiments, theplurality of moieties are covalently attached to the polymer molecules.In various embodiments, each of the plurality of moieties may beattached to a separate polymer molecule, or several of the plurality ofmoieties may be attached to the same polymer molecule.

As an example embodiment, a composition is provided comprising(S)-(−)-nicotine or a metabolite thereof conjugated to a polymer. Inpreferred embodiments, the conjugate is via a covalent linkage. Thepolymer may be any of the polymers described herein.

For example, in some embodiments the polymer is a biocompatible,biodegradable polymer. In some embodiments, the polymer is ahomopolymer, while in other embodiments, the polymer is a copolymer. Forexample, the polymer is a linear copolymer comprising a hydrophobicblock with a hydrophobic terminus and a hydrophilic block with acorresponding hydrophilic terminus. For example, the polymer is acopolymer comprising blocks selected from poly(lactic acid), derivativesof poly(lactic acid), PEGylated poly(lactic acid),poly(lactic-co-glycolic acid), derivatives of poly(lactic-co-glycolicacid), PEGylated poly(lactic-coglycolic acid), poly(anhydrides),PEGylated poly(anhydrides), poly(ortho esters) derivatives of poly(orthoesters), PEGylated poly(ortho esters), poly(caprolactones), derivativesof poly(caprolactone), PEGylated poly(caprolactones), polylysine,derivatives of polylysine, PEGylated polylysine, poly(ethylene imine),derivatives of poly(ethylene imine), PEGylated poly(ethylene imine),poly(acrylic acid), derivatives of poly(acrylic acid), PEGylatedpoly(acrylic acid), poly(urethane), PEGylated poly(urethane),derivatives of poly(urethane), and combinations thereof.

In some embodiments, a plurality of nicotine moieties are conjugated tothe polymer, while in other embodiments, a single nicotine moiety isconjugated to the polymer. In the former case, the nicotine moieties maybe located as sidechains attached to the polymer backbone, or thepolymer may comprise a plurality of endgroups (as in, for example, adendritic or branched polymer), a plurality of which are attached to anicotine moiety. In the latter case, the nicotine moiety may be locatedat any point along the backbone of the polymer. In preferredembodiments, the nicotine moiety is located at one terminus of thepolymer. For example, in the case of an amphiphilic polymer having ahydrophobic block with a hydrophobic terminus and a hydrophilic blockwith a hydrophilic terminus, the nicotine moiety may be covalentlyattached at the hydrophilic or hydrophobic terminus. For example, thenicotine moiety is located at the hydrophilic terminus.

Nicotine comprises a pyridine ring and a pyrrolidine ring, and thecovalent linkage to the polymer may be at the 2-, 4-, 5-, or 6-positionof the pyridine ring, or the 3-, 4-, or 5-position of the pyrrolidinering. For example, the composition may comprise a conjugate comprisingnicotine or a metabolite thereof conjugated to a polymer. For example,the polymer conjugate may have the structure of formula (I)

(X)_(n)-L1-(Y)_(m)-L2-A  (I)

wherein:

X is a hydrophobic polymer segment;

Y is a hydrophilic polymer segment;

n and m are selected from 0 and 1, provided that n and m are not both 0;

L1 and L2 are independently selected from a bond and a linking group;and

A is (S)-(−)-nicotine.

For example, A may have the structure

wherein one of R¹-R⁷ is (X)_(n)-L1-(Y)_(m)-L2-, and the others areindependently selected from H, alkyl, aryl, alkoxy, aryloxy, alkaryl,and aralkyl, any of which may be substituted or unsubstituted and maycontain one or more heteroatoms. When A is a nicotine metabolite (suchas any of those described herein), the linkage to (X)_(n)-L1-(Y)_(m)-L2-may be at any available position on the metabolite, similar to nicotineas shown above.

Also for example, L1 and L2 may be selected from substituted andunsubstituted C₁-C₃₀ hydrocarbylene, C₁-C₃₀ halocarbylene, heteroatoms,and functional groups. For example, such linker moieties includesubstituted and unsubstituted C₁-C₃₀ alkylene, C₂-C₃₀ alkenylene, C₂-C₃₀alkynylene, C₅-C₃₀ arylene, C₆-C₃₀ alkenylene, and C₆-C₃₀ aralkylene,any of which may be heteroatom-containing and/or halogenated (including,for example, perfluorinated or perchlorinated). Functional groups andheteroatoms suitable as linker moieties include thioether (—S—), ether(—O—), amino (—NR—), ketone (—C(═O)—), imino (—C(═NR)—), amido(—C(═O)—NR—), ester (—(C═O)—O—, also referred to as carboalkoxy),disulfide (—S—S—), carbonato (—O—C(═O)-0), carbodiimido (—N═C═N—),carbamate (—NH—C(═O)—O—), and the like.

For example, a composition comprising a conjugate according to formula(I), where L1 and L2 are both bonds, n and m are both 1, X is a PLAsegment, and Y is a PEG segment may have the structure

wherein p and q are both integers.

The nicotine-polymer conjugates previously described may, in someembodiments, self-assemble into nanoparticles (herein also referred toas “nicotine nanoparticles” or “nicotine-NP”). Amphiphilic polymers areparticularly well suited for such self-assembly. Depending upon theconditions of formation, self-assembled nicotine nanoparticles formed inthis fashion may have any of the characteristics described herein (e.g.,particle size, distribution, hydrophobicity/hydrophilicity of the coreand exterior, etc.).

In some embodiments, amphiphilic polymer-nicotine conjugates having thestructure of formula (I), where n and m are both equal to 1, are used toprepare nicotine nanoparticles wherein the core is hydrophobic and theperiphery is hydrophilic. The nicotine nanoparticles may furthercomprise amphiphilic copolymers not conjugated to nicotine; suchpolymers may, for example, have the structure (X)_(n)-L1-(Y)_(m),wherein X, Y, L1, n, and m are as defined above.

The nicotine nanoparticles may further comprise an immunostimulatoryagent, such as any of those described herein. For example, the nicotinenanoparticles may comprise an adjuvant. The adjuvant may, in someembodiments, be conjugated to the amphiphilic polymers. Alternatively orin addition, the adjuvant may be conjugated to a homopolymer formed froma hydrophobic polymer of from a hydrophilic polymer. Alternatively or inaddition, the adjuvant may be present in free form. In some preferredembodiments, a combination of free adjuvant and adjuvant conjugated to ahydrophobic polymer (i.e., any of the polymers suitable for X in formula(I)) is encapsulated within the core of the nicotine nanoparticles(i.e., the adjuvant is not covalently attached to the amphiphilicpolymers that form the nanoparticles).

The nicotine nanoparticles may further comprise one or more targetingmoieties such as any of those targeting moieties described herein. Forexample, the nicotine nanoparticles may comprise a subcapsular sinusmacrophage (SCS-Mph) targeting moiety.

Each nicotine nanoparticle comprises a plurality of nicotine-polymerconjugate molecules, so that each nanoparticle comprises a plurality ofnicotine moieties. Typically, where the nicotine moieties are conjugatedto the hydrophilic block, the nicotine moieties tend to concentrate inthe periphery of the particles, and particularly at the surface of theparticles.

Various characteristics of the nicotine nanoparticles can be controlledby modifying the composition. One example is the location within thenanoparticles of the nicotine moieties and of the adjuvant (whenadjuvant is present). Also for example, release kinetics of encapsulatedadjuvant can be modified. Such modifications can be illustrated using,for example, nicotine nanoparticles comprising one or more of thefollowing components: an amphiphilic copolymer (“X—Y”); anicotine-amphiphilic copolymer conjugate (“X—Y-nicotine”); an adjuvant;and an adjuvant-homopolymer conjugate (“adjuvant-X”). Adjustments in therelative proportion of the various components have the effect ofmodifying the structure of the composition. For example, the percentageof X—Y relative to X—Y-nicotine can be modified to affect the density ofnicotine moieties on the nanoparticle surface. The molecular weight ofthe hydrophobic blocks (i.e., “X”) relative to the hydrophilic blocks(i.e., “Y”) can be modified to affect the size of the particle corerelative to the size of the particle periphery. Release kinetics for theadjuvant can be modified by adjusting the amount of free adjuvant andadjuvant-X. Such modifications to control the characteristics of thenicotine nanoparticles are further illustrated in the Examples sectionprovided herein.

It will be appreciated that the foregoing example of nicotine-polymerconjugates for forming nanoparticles is provided merely for the purposeof illustration, not limitation. Variations of the example according toany of the disclosure provided herein are within the scope of thepresent invention. For example, as stated earlier, the term “nicotine”is meant to include derivatives, analogs, and metabolites of nicotine.Accordingly, the vaccine nanocarriers described above may comprise aderivative, analog, or metabolite of nicote (rather than nicotine perse). For example, the nanocarriers may comprise a polymer covalentlyconjugated to a metabolite of nicotine (such as cotinine, etc.). Forexample, in formula (I) provided above, A may be a metabolite,derivative, or analog of nicotine.

Additional examples of derivatives of nicotine can be found, forexample, in U.S. Pat. Nos. 6,232,082 (to Ennifar et al.) and 6,932,971(to Bachmann et al.), the relevant portions of the disclosures of whichare incorporated herein by reference.

As previously stated, nicotine haptens suitable for the conjugates ofthe present invention can have at least one, preferably one, polymerchain bonded to any position on either the pyridine or the pyrrolidinering of the nicotine. For example, nicotine may be chemicallyderivatized at the 3′ position to provide an hydroxyl residue that issuitable for reaction with reagents such as succinic anhydride to formO-succinyl-3′-hydroxymethyl-nicotine. This nicotine derivative may becoupled to amino acids of the core particle, such as lysine, using theactivation reagent EDC. In a further preferred embodiment theO-succinyl-3′-hydroxymethyl-nicotine can be activated with EDC and theresulting activated carboxylic group is stabilized byN-hydroxysuccinimide. In other embodiments, nicotine derivatives areproduced by acylation of nornicotine with succinic anhydride inmethylene chloride in the presence of two equivalents ofdiisopropylethylamine. Such a nicotine hapten is then coupled to coreparticles of present invention with an activating reagent e.g. HATU. Inone embodiment, the precursor of the conjugates is synthesized byacylating racemic nornicotine with succinic anhydride in methylenechloride in the presence of two equivalents of diisopropylethylamine.The product of this reaction is then coupled to the lysine residue of acarrier protein using HATU to obtain the conjugate. In anotherembodiment, selectively alkylating the pyridine nitrogen in(S)-(−)-nicotine in anhydrous methanol, with ethyl 3-bromobutyrate,5-bromovaleric acid, 6-bromohexanoic acid or 8-bromooctanoic acid yieldproducts suitable for conjugation to a carrier protein using HATU.

For example, in one embodiment, 6-(carboxymethylureido)-(±)-nicotine(CMUNic) conjugate is synthesized from 6-amino-(±)-nicotine, which isreacted with ethyl isocyanoacetate to form6-(carboxyethylureido)-(±)-nicotine, and hydrolysis by lithium hydroxideto form CMUNic. The hapten is conjugated via the terminal carboxylgroup, which may be activated using e.g.1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl. In anotherembodiment, 6-amino-(±)-nicotine is coupled according to the invention.In another embodiment of the present invention,trans-3′-aminomethylnicotine conjugate is prepared bytrans-3′-hydroxymethylnicotine alcohol via the tosylate. The hapten isconjugated through a succinic acid linker using1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl (EDAC) to activatethe linker's carboxylic acid group. In a related embodiment, 3′-linkagesto nicotine haptens are performed by first generatingtrans-3′-hydroxymethylnicotine which is reacted with succinic anhydrideto yield the succinylated hydroxymethylnicotine(O-succinyl-3′-hydroxymethyl-nicotine). This product is then mixed withEDAC and, for example, a suitably functionalized polymer forcarbodiimide-activated coupling. In another embodiment,trans-4′-carboxycotinine is similarly activated with EDAC for coupling.

In one embodiment, a nicotine hapten is coupled via the 1-positionNitrogen by conversion to the aminoethylpyridinium derivative,S-1-(b-aminoethyl)nicotinium chloride dihydrochloride, which is thencoupled to, for example, a suitably functionalized polymer in thepresence of 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimidemetho-p-toluenesulfonate. In a related embodiment, Cotinine isconjugated to polymers using the same general approach, via formation ofS-1-(b-aminoethyl) cotinium chloride hydrochloride. In one embodiment, anicotine hapten is coupled via the 1′-position via formation ofN-[1-oxo-6-[(2S)-2-(3-pyridyl)-1-pyrrolidinyl]hexyl]-β-alanine. Thisactivated hapten is then coupled to a suitably functionalized polymer.In three other embodiments, conjugates are formed using cotinine hapten4-oxo-4-[[6-[(5S)-2-oxo-5-(3-pyridinyl)-1-pyrrolidinyl]]hexyl]amino]-butanoicacid, or the nomicotine haptens(2S)-2-(3-pyridinyl)-1-pyrrolidinebutanoic acid phenylmethyl ester or(2R)-2-(3-pyridinyl)-1-pyrrolidinebutanoic acid phenylmethyl ester. Inone embodiment, cotinine 4′-carboxylic acid is covalently bound to, forexample, a suitably functionalized polymer. Nicotine haptens may also beconjugated via a linker at the 6-position of nicotine. Along theselines, the following haptens are used in embodiments of the presentinvention N-succinyl-6-amino-(±)-nicotine;6-(σ-aminocapramido)-(±)-nicotine and 6-(σ-aminocapramido)-(±)-nicotine.In other embodiments of the invention, nicotine haptens are conjugatedvia the 3′, 4′, or 5′ position via succinylation of aminomethylnicotine,activation with EDC and subsequent mixture with the conjugate. In otherembodiments, aminomethyl nicotine is conjugated via polyglutamicacid-ADH to the conjugate. In other embodiments, conjugates are formedfrom acetyl nicotine and aldehydro nicotine derivatized at the 3′, 4′,or 5′ positions. In other embodiments, hapten carrier conjugatescomprise 5- and 6-linkages of nicotine, including5-(1-methyl-2-pyrrolidinyl)-2- or 3-pyridinyl-conjugates and5-(N-methyl-2-pyrrolidinyl)-2- or 3-pyridinyl-conjugates. In otherembodiments, 5- and 6-amino nicotine are utilized as starting materialsthat are further derivatized at the amino group to add, typically,carbon chains that terminate in a suitably reactive group includingamines and carboxylic acids. These haptens are then suitable forconjugation. In other embodiments, 5- or 6-bromonicotine is used as asuitable starting material for reaction with alkynes leading to theaddition of unsaturated carbon groups with a chain which terminate withmoeities suitable for coupling, including amines and carboxylic acids,that allow conjugation. Other embodiments of the present inventioncomprise conjugates comprising nicotine haptens conjugated at the 1, 2,4, 5, 6, or 1′ positions of the nicotine.

It will be appreciated that the compounds named above can be used eitheras racemic mixtures of enantiomers or as the pure enantiomer (in anyconfiguration). It will further be appreciated that the nanocarriers ofthe invention may comprise a combination of different nicotinederivatives/analogs/metabolites (including nicotine per se).

Applications

The compositions and methods described herein can be used to induce,enhance, suppress, direct, or redirect an immune response. Thecompositions and methods described herein can be used for theprophylaxis and/or treatment of any cancer, infectious disease,metabolic disease, degenerative disease, autoimmune disease,inflammatory disease, immunological disease, or other disorder and/orcondition. The compositions and methods described herein can also beused for the treatment of an addiction, such as an addiction to any ofthe addictive substances described herein. The compositions and methodsdescribed herein can also be used for the prophylaxis and/or treatmentof a condition resulting from the exposure to a toxin, hazardoussubstance, environmental toxin, or other harmful agent. Subjectsinclude, but are not limited to, humans and/or other primates; mammals,including commercially relevant mammals such as cattle, pigs, horses,sheep, cats, and/or dogs; and/or birds, including commercially relevantbirds such as chickens, ducks, geese, and/or turkeys.

In some embodiments, vaccine nanocarriers in accordance with the presentinvention may be used to treat, alleviate, ameliorate, relieve, delayonset of, inhibit progression of, reduce severity of, and/or reduceincidence of one or more symptoms or features of a disease, disorder,and/or condition. In some embodiments, inventive vaccine nanocarriersmay be used to treat, alleviate, ameliorate, relieve, delay onset of,inhibit progression of, reduce severity of, and/or reduce incidence ofone or more symptoms or features of microbial infection (e.g. bacterialinfection, fungal infection, viral infection, parasitic infection,etc.).

In one aspect of the invention, a method for the prophylaxis and/ortreatment of a disease, disorder, or condition (e.g., a microbialinfection) is provided. In some embodiments, the prophylaxis and/ortreatment of the disease, disorder, or condition comprises administeringa therapeutically effective amount of inventive vaccine nanocarriers toa subject in need thereof, in such amounts and for such time as isnecessary to achieve the desired result. In certain embodiments of thepresent invention a “therapeutically effective amount” of an inventivevaccine nanocarrier is that amount effective for treating, alleviating,ameliorating, relieving, delaying onset of, inhibiting progression of,reducing severity of, and/or reducing incidence of one or more symptomsor features of microbial infection. In some embodiments, a“therapeutically effective amount” is an amount effective to modulatethe immune system. Such an amount may be an immunogenic amount, i.e., anamount sufficient to elicit a detectable immune response in a subject,e.g., a detectable antibody response and/or detectable T cell response.

Inventive prophylactic and/or therapeutic protocols involveadministering a therapeutically effective amount of one or moreinventive vaccine nanocarriers to a healthy subject (e.g., a subject whodoes not display any symptoms of microbial infection and/or who has notbeen diagnosed with microbial infection; a subject who has not yet beenexposed to a toxin, a subject who has not yet ingested an abused oraddictive substance, etc.). For example, healthy individuals may bevaccinated using inventive vaccine nanocarrier(s) prior to developmentof microbial infection, exposure to the toxin, abused substance,addictive substance, etc. and/or onset of symptoms related thereto; atrisk individuals (e.g., patients exposed to individuals suffering frommicrobial infection, traveling to locations where microbes/toxins areprevalent; etc.) can be treated substantially contemporaneously with(e.g., within 48 hours, within 24 hours, or within 12 hours of) theonset of symptoms of and/or exposure/ingestion. Of course individualsknown to have microbial infection, have been exposed to a toxin, oringested an abused or additive substance may receive treatment at anytime.

In some embodiments, inventive prophylactic and/or therapeutic protocolsinvolve administering a therapeutically effective amount of one or moreinventive vaccine nanocarriers to a subject such that an immune responseis stimulated in both T cells and B cells.

In some embodiments, by combining selected immunomodulatory agents withtargeting moieties and immunostimulatory agents for different APCs,immune responses (e.g. effector responses) can be tailored topreferentially elicit the most desirable type of immune response for agiven indication, e.g., humoral response, type 1 T cell response, type 2T cell response, cytotoxic T cell, response, and/or a combination ofthese responses. Thus, the same platform may be used for a broad rangeof different clinical applications, including prophylactic vaccines to ahost of pathogens as well as immunotherapy of existing diseases, such asinfections, autoimmune diseases, and/or cancer.

Cancers include but are not limited to biliary tract cancer; braincancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer;endometrial cancer; esophageal cancer; gastric cancer; intraepithelialneoplasms; lymphomas; liver cancer; lung cancer (e.g., small cell andnon-small cell); melanoma; neuroblastomas; oral cancer; ovarian cancer;pancreatic cancer; prostate cancer; rectal cancer; sarcomas; skincancer; testicular cancer; thyroid cancer; and renal cancer, as well asother carcinomas and sarcomas.

Autoimmune diseases include, but are not limited to, rheumatoidarthritis, rheumatic fever, ulcerative colitis, celiac disease, Crohn'sdisease, inflammatory bowel disease, insulin-dependent diabetesmellitus, diabetes mellitus, juvenile diabetes, spontaneous autoimmunediabetes, gastritis, autoimmune atrophic gastritis, autoimmunehepatitis, thyroiditis, Hashimoto's thyroiditis, autoimmune thyroiditis,insulitis, oophoritis, orchitis, uveitis, phacogenic uveitis, multiplesclerosis, myasthenia gravis, primary myxoedema, thyrotoxicosis,pernicious anemia, autoimmune haemolytic anemia, Addison's disease,scleroderma, Goodpasture's syndrome, Guillain-Barre syndrome, Graves'disease, glomerulonephritis, psoriasis, pemphigus vulgaris, pemphigoid,sympathetic opthalmia, idiopathic thrombocylopenic purpura, idiopathicleucopenia, Siogren's syndrome, Wegener's granulomatosis,poly/dermatomyositis or systemic lupus erythematosus.

Inflammatory disease/disorders include, for example, cardiovasculardisease, chronic obstructive pulmonary disease (COPD), bronchiectasis,chronic cholecystitis, tuberculosis, Hashimoto's thyroiditis, sepsis,sarcoidosis, silicosis and other pneumoconioses, and an implantedforeign body in a wound, but are not so limited. As used herein, theterm “sepsis” refers to a well-recognized clinical syndrome associatedwith a host's systemic inflammatory response to microbial invasion. Theterm “sepsis” as used herein refers to a condition that is typicallysignaled by fever or hypothermia, tachycardia, and tachypnea, and insevere instances can progress to hypotension, organ dysfunction, andeven death.

In some embodiments, the present invention is directed to nanoparticlecompositions suitable for eliciting an immune response.

In some embodiments, the present invention is directed tonanoparticle-nicotine bioconjugates that comprise: (i) a nanoparticlecapable of carry an adjuvant (encapsulated and/or surfacefunctionalized) to the cell surface of Antigen Presenting Cells (APCs);(ii) one or more molecules on a surface of the nanoparticle to targetspecific cells; and (iii) nicotine moieties that are capable ofeliciting an immunogenic response. In some embodiments, the chirality ofthe nicotine moieties is controlled when covalently attached to apolymer and at a surface of the nanoparticles.

In some embodiments, the present invention is directed to enhancing thepotentiating of an immune response in a mammal, comprising administeringan effective amount of a nanoparticle-nicotine bioconjugate of thepresent invention to enhance the immune response of a mammal to one ormore antigens. The present invention is also directed to a method ofvaccination of a mammal.

Thus, in some embodiments, the nanoparticle-nicotine conjugates areeffective to provide an increased antibody concentration in a subject,particularly an increased IgG response to nicotine. Such conjugates arefurthermore capable of targeting SCS Mphs, thereby invoking a humoralimmune response.

In some embodiments, the present invention provides novelnanoparticle-nicotine bioconjugates that are stable, employ a nicotinederivative with a linkage that preserves the nature of the nicotineepitope(s), and the relative orientation of the two rings of thenicotine molecule in its natural (S)-(−) formation and/or (R)-(−). Bothrings of nicotine, and their relative orientation, are believed to beessential for the recognition by antibody of nicotine in solution. Insome embodiments, the invention includes nanoparticle-nicotinebioconjugates wherein the nicotine derivative is conjugated from anyposition of the nicotine.

Such conjugates are capable of stimulating the production of antibodiesthat are capable of specifically binding to nicotine. In preferredembodiments, upon injection into a mammal, a given dose ofnanoparticle-nicotine bioconjugate formulation with adjuvant willstimulate immune cells (particularly APCs, including dendritic cells(DCs), macrophages and B cells or any combination of these APCs) morepotently than the same dose of nanoparticle adjuvant formulationadministered with free nicotine derivatives. The nanoparticle-nicotineadjuvant stimulated APCs are thus enabled to promote more vigorousinnate and adaptive immune responses to antigens, including immuneresponses by B cells and T cells.

One utility of the nanoparticle-nicotine bioconjugate lies in theenhanced induction or conversion of immune responses to therapeutic andprophylactic vaccines by a given quantity nanoparticle presentingnicotine derivatives with a controlled chiral composition on thesurface. This enhanced efficacy, especially when combined with theconcept of targeting nanoparticles to specific APCs, can substantiallyreduce the amount or frequency at which vaccines must be administered toachieve a desired response, resulting in a substantially decreased riskof toxicity and off-target effects.

Another utility is that encapsulation and controlled/triggered releaseof adjuvant will result in reduced pleiotropic effects on bystandercells, thus affording enhanced safety by reducing the risk of undesiredside effects and allowing administration of larger adjuvant doses formaximal stimulation of therapeutic and prophylactic immune responses.

In some embodiments, the present invention provides a method of treatingnicotine addiction by administering a nanoparticle bioconjugate to apatient addicted to nicotine, thereby generating anti-nicotineantibodies in the patient. Thus, when the patient smokes (or useschewing tobacco), the nicotine from these products will be bound by theanti-nicotine antibodies in the blood, preventing the nicotine fromcrossing the blood-brain barrier. This significantly reduces oreliminates nicotine-induced alterations in brain chemistry, which is thesource of nicotine-addiction. A further advantage of using nanoparticlesis the ability to design the carrier for long-circulation period in theblood. In this regard, it is important that the nicotine-carrierconjugate elicit the production of antibodies that will recognizenicotine molecule(s).

Nanoparticle carriers, in contrast from currently prevalent strategies,have several major advantages: 1. nanoparticle-nicotine conjugatesprovide recognition template for anti-nicotine antibodies; 2. relativelysimple scale-up production and purification of nanoparticle-nicotinebioconjugates; 3. desirable long-circulating properties in blood; 4.controlled delivery of adjuvant leading to minimal off-target action andresulting in reduced side effects; 5. enhanced targeting to APCs leadingto enhanced therapeutic and prophylactic immune responses to vaccines;and 6. reduced toxicity allows the safe administration of larger dosesof nanoparticle-nicotine bioconjugates to maximize immune responses.

In some embodiments, the present invention encompasses therapeuticmethods that prevent nicotine from crossing the blood brain barrier. Inparticular, administration of nanoparticle-nicotine bioconjugates to apatient will generate antibodies against nicotine and its metabolites,in the bloodstream of the patient. Alternatively, antinicotineantibodies generated outside the body of the patient to be treated, in asuitable host mammal, can be administered to a patient. If the patientsmokes, the nicotine in his/her blood will be bound by the circulatinganti-nicotine antibodies, preventing the nicotine from reaching thebrain. Therefore, the antibodies will prevent the physiological andpsychological effects of nicotine that originate in the brain. Becausethe smoker will experience a lessening or cessation of these effects,he/she will lose the desire to smoke. The same therapeutic effects areexpected if a patient uses smokeless tobacco, after being immunized witha nanoparticle-nicotine bioconjugates of the invention. Additionally,the conjugates and antibodies of the invention may exert their effectsby affecting the ability of nicotine to stimulate the peripheral nervoussystem.

In some embodiments, the conjugates of the invention are suitable fortreating and preventing nicotine addiction. For treating nicotineaddiction, a nanoparticle-nicotine bioconjugate of the invention isadministered to a patient suffering from nicotine addiction. Forpreventing nicotine addiction, patients at risk for developing nicotineaddiction, such as teenagers, are treated with a conjugate according tothe invention. Direct administration of the conjugate to a patient maybe referred to as “active immunization.”

A vaccine composition of the present invention comprises at least onenanoparticle-nicotine bioconjugates in an amount sufficient to elicit animmune response thereto. Initial vaccination with thenanoparticle-nicotine bioconjugate of the present invention creates hightiters of antibodies that are specific to nicotine. The therapeuticallyeffective amount of nanoparticle-nicotine bioconjugate which isadministered to a patient in need of treatment for nicotine addiction isreadily determined by the skilled artisan. Suitable dosage ranges are1-1000 ng/dose. It generally takes a patient one to several weeks togenerate antibodies against a foreign antigen. The production ofantibodies in a patient's blood can be monitored by using techniquesthat are well-known to the skilled artisan, such as ELISA,radioimmunoassay, and Western blotting methods. Therapeuticeffectiveness also can be monitored by assessing various physicaleffects of nicotine, such as blood pressure. The inventivenanoparticle-nicotine bioconjugate can be processed to afford acomposition which can be readily administered to a patient. Thepreferred modes of administration include but are not limited tointranasal, intratracheal, oral, dermal, transmucosal subcutaneousinjection and intravenous injection. The skilled artisan will recognizethat the initial injection may be followed by subsequent administrationof one or more “boosters” of conjugate. Such a booster will increase theproduction of antibodies against the nanoparticle-nicotine bioconjugateof the invention. Also as described herein, the nanoparticle nicotinecompositions of the present invention may optionally contain one or morepharmaceutically acceptable excipients. The excipients useful in thepresent include those described herein, such as sterile water, saltsolutions such as saline, sodium phosphate, sodium chloride, alcohol,gum arabic, vegetable oils, benzyl alcohols, polyethylene glycol,gelatin, mannitol, carbohydrates, magnesium stearate, viscous paraffin,fatty acid esters, hydroxy methyl cellulose and buffers.

As described herein, the nanoparticle-nicotine bioconjugate of thepresent invention, in order to be administered to a patient in need oftreatment or prevention of nicotine addiction, are incorporated into apharmaceutical composition. When the composition containing thenanoparticle-nicotine bioconjugate is to be used for injection, it ispreferable to deliver nanoparticle-nicotine bioconjugate in an aqueous,saline solution at a pharmaceutically acceptable pH. However, it ispossible to use an injectable suspension of nanoparticle-nicotinebioconjugate. In addition to the usual pharmaceutically acceptableexcipients, the composition may contain optional components to ensurepurity, enhance bioavailability and/or increase penetration. Thepharmaceutical compositions of the present invention are preferablyprepared in a sterile formulation and are sufficiently stable towithstand storage, distribution, and use. Additionally, the compositionmay contain additional components in order to protect the compositionfrom infestation with, and growth of, microorganisms. In some preferredembodiments, the composition is manufactured in the form of alyophilized powder which is to be reconstituted by a pharmaceuticallyacceptable diluent just prior to administration. Methods of preparingsterile injectable solutions are well known to the skilled artisan andinclude, but are not limited to, vacuum drying, freeze-drying, and spindrying. These techniques yield a powder of the active ingredient alongwith any additional components.

As described previously, the nanoparticle nicotine conjugatepharmaceutical compositions of this invention can be administered to apatient by any means known in the art including orally, in an aerosol,or via topical or parenteral routes. In certain embodiments parenteralroutes are preferred since they avoid contact with the digestive enzymesthat are found in the alimentary canal. According to such embodiments,inventive compositions may be administered by injection (e.g.,intravenous, subcutaneous or intramuscular, intraperitoneal injection),rectally, vaginally, topically (as by powders, creams, ointments, ordrops), or by inhalation (as by sprays), oral, intrapulmonary,intrabiliary, intravenously, and intranasal. For oral administration,the pharmaceutical compositions may take the form of, for example,tablets or capsules prepared by conventional means with pharmaceuticallyacceptable excipients such as binding agents (e.g., pregelatinised maizestarch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers(e.g., lactose, microcrystalline cellulose or calcium hydrogenphosphate); lubricants (e.g., magnesium stearate, talc or silica);disintegrants (e.g., potato starch or sodium starch glycolate); orwetting agents (e.g., sodium lauryl sulphate). The tablets may be coatedby methods well known in the art. Liquid preparations for oraladministration may take the form of, for example, solutions, syrups orsuspensions, or they may be presented as a dry product for constitutionwith water or other suitable vehicle before use. Such liquidpreparations may be prepared by conventional means with pharmaceuticallyacceptable additives such as suspending agents (e.g., sorbitol syrup,cellulose derivatives or hydrogenated edible fats); emulsifying agents(e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oilyesters, ethyl alcohol or fractionated vegetable oils); and preservatives(e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). Thepreparations may also contain buffer salts, flavoring, coloring andsweetening agents as appropriate. Preparations for oral administrationmay be suitably formulated to give controlled release of the activecompound. For buccal or sublingual administration, the compositions maytake the form of tablets or lozenges formulated in conventional manner.For rectal and vaginal routes of administration, the active ingredientmay be formulated as solutions (for retention enemas) suppositories orointments. For administration by inhalation, the active ingredient canbe conveniently delivered in the form of an aerosol spray presentationfrom pressurized packs or a nebulizer, with the use of a suitablepropellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g. gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch. For prolonged delivery, the active ingredient canbe formulated as a depot preparation, for administration byimplantation; e.g., subcutaneous, intradermal, or intramuscularinjection. Thus, for example, the active ingredient may be formulatedwith suitable polymeric or hydrophobic materials (e.g., as an emulsionin an acceptable oil) or ion exchange resins, or as sparingly solublederivatives; e.g., as a sparingly soluble salt form. The present vaccinecomposition can be used in combination with compounds or other therapiesthat are useful in the treatment of addiction.

Pharmaceutical Compositions

The present invention provides novel compositions comprising atherapeutically effective amount of one or more vaccine nanocarriers andone or more pharmaceutically acceptable excipients. In some embodiments,the present invention provides for pharmaceutical compositionscomprising inventive vaccine nanocarriers and/or any of the compositionsthereof described herein. Such pharmaceutical compositions mayoptionally comprise one or more additional therapeutically-activesubstances. In accordance with some embodiments, a method ofadministering a pharmaceutical composition comprising inventivecompositions to a subject in need thereof is provided. In someembodiments, inventive compositions are administered to humans. For thepurposes of the present invention, the phrase “active ingredient”generally refers to an inventive vaccine nanocarrier comprising at leastone immunomodulatory agent and optionally comprising one or moretargeting moieties, immunostimulatory agents, and/or nanoparticles.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and/or perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and/or other primates; mammals, includingcommercially relevant mammals such as cattle, pigs, horses, sheep, cats,and/or dogs; and/or birds, including commercially relevant birds such aschickens, ducks, geese, and/or turkeys.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmaceutics. In general, such preparatory methods include the step ofbringing the active ingredient into association with one or moreexcipients and/or one or more other accessory ingredients, and then, ifnecessary and/or desirable, shaping and/or packaging the product into adesired single- or multi-dose unit.

A pharmaceutical composition of the invention may be prepared, packaged,and/or sold in bulk, as a single unit dose, and/or as a plurality ofsingle unit doses. As used herein, a “unit dose” is discrete amount ofthe pharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject and/or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient(s), and/or any additional ingredients in apharmaceutical composition of the invention will vary, depending uponthe identity, size, and/or condition of the subject treated and furtherdepending upon the route by which the composition is to be administered.By way of example, the composition may comprise between 0.1% and 100%(w/w) active ingredient.

Pharmaceutical formulations of the present invention may additionallycomprise a pharmaceutically acceptable excipient, which, as used herein,includes any and all solvents, dispersion media, diluents, or otherliquid vehicles, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, solidbinders, lubricants and the like, as suited to the particular dosageform desired. Remington's The Science and Practice of Pharmacy, 21^(st)Edition, A. R. Gennaro, (Lippincott, Williams & Wilkins, Baltimore, Md.,2006; incorporated herein by reference) discloses various excipientsused in formulating pharmaceutical compositions and known techniques forthe preparation thereof. Except insofar as any conventional excipient isincompatible with a substance or its derivatives, such as by producingany undesirable biological effect or otherwise interacting in adeleterious manner with any other component(s) of the pharmaceuticalcomposition, its use is contemplated to be within the scope of thisinvention.

In some embodiments, the pharmaceutically acceptable excipient is atleast 95%, 96%, 97%, 98%, 99%, or 100% pure. In some embodiments, theexcipient is approved for use in humans and for veterinary use. In someembodiments, the excipient is approved by United States Food and DrugAdministration. In some embodiments, the excipient is pharmaceuticalgrade. In some embodiments, the excipient meets the standards of theUnited States Pharmacopoeia (USP), the European Pharmacopoeia (EP), theBritish Pharmacopoeia, and/or the International Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, dispersing and/or granulating agents, surface active agentsand/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils. Suchexcipients may optionally be included in the inventive formulations.Excipients such as cocoa butter and suppository waxes, coloring agents,coating agents, sweetening, flavoring, and perfuming agents can bepresent in the composition, according to the judgment of the formulator.

Exemplary diluents include, but are not limited to, calcium carbonate,sodium carbonate, calcium phosphate, dicalcium phosphate, calciumsulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose,cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc.,and combinations thereof.

Exemplary granulating and/or dispersing agents include, but are notlimited to, potato starch, corn starch, tapioca starch, sodium starchglycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite,cellulose and wood products, natural sponge, cation-exchange resins,calcium carbonate, silicates, sodium carbonate, cross-linkedpolyvinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch(sodium starch glycolate), carboxymethyl cellulose, cross-linked sodiumcarboxymethyl cellulose (croscarmellose), methylcellulose,pregelatinized starch (starch 1500), microcrystalline starch, waterinsoluble starch, calcium carboxymethyl cellulose, magnesium aluminumsilicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds,etc., and combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are notlimited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodiumalginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin,egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidalclays (e.g. bentonite [aluminum silicate] and Veegum [magnesium aluminumsilicate]), long chain amino acid derivatives, high molecular weightalcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetinmonostearate, ethylene glycol distearate, glyceryl monostearate, andpropylene glycol monostearate, polyvinyl alcohol), carbomers (e.g.carboxy polymethylene, polyacrylic acid, acrylic acid polymer, andcarboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g.carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylenesorbitan monolaurate [Tween® 20], polyoxyethylene sorbitan [Tween® 60],polyoxyethylene sorbitan monooleate [Tween® 80], sorbitan monopalmitate[Span® 40], sorbitan monostearate [Span® 60], sorbitan tristearate[Span® 65], glyceryl monooleate, sorbitan monooleate [Span® 80]),polyoxyethylene esters (e.g. polyoxyethylene monostearate [Myrj® 45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and Solutol), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g. Cremophor®), polyoxyethyleneethers, (e.g. polyoxyethylene lauryl ether [Brij® 30]),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include, but are not limited to, starch (e.g.cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose,dextrose, dextrin, molasses, lactose, lactitol, mannitol,); natural andsynthetic gums (e.g. acacia, sodium alginate, extract of Irish moss,panwar gum, ghatti gum, mucilage of isapol husks,carboxymethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate,poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum), and larcharabogalactan); alginates; polyethylene oxide; polyethylene glycol;inorganic calcium salts; silicic acid; polymethacrylates; waxes; water;alcohol; etc.; and combinations thereof.

Exemplary preservatives may include antioxidants, chelating agents,antimicrobial preservatives, antifungal preservatives, alcoholpreservatives, acidic preservatives, and other preservatives. Exemplaryantioxidants include, but are not limited to, alpha tocopherol, ascorbicacid, acorbyl palmitate, butylated hydroxyanisole, butylatedhydroxytoluene, monothioglycerol, potassium metabisulfite, propionicacid, propyl gallate, sodium ascorbate, sodium bisulfite, sodiummetabisulfite, and sodium sulfite. Exemplary chelating agents includeethylenediaminetetraacetic acid (EDTA), citric acid monohydrate,disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malicacid, phosphoric acid, sodium edetate, tartaric acid, and trisodiumedetate. Exemplary antimicrobial preservatives include, but are notlimited to, benzalkonium chloride, benzethonium chloride, benzylalcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine,chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol,glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethylalcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.Exemplary antifungal preservatives include, but are not limited to,butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoicacid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodiumbenzoate, sodium propionate, and sorbic acid. Exemplary alcoholpreservatives include, but are not limited to, ethanol, polyethyleneglycol, phenol, phenolic compounds, bisphenol, chlorobutanol,hydroxybenzoate, and phenylethyl alcohol. Exemplary acidic preservativesinclude, but are not limited to, vitamin A, vitamin C, vitamin E,beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbicacid, sorbic acid, and phytic acid. Other preservatives include, but arenot limited to, tocopherol, tocopherol acetate, deteroxime mesylate,cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened(BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ethersulfate (SLES), sodium bisulfite, sodium metabisulfite, potassiumsulfite, potassium metabisulfite, Glydant Plus®, Phenonip®,methylparaben, Germall 115, Germaben II, Neolone™, Kathon™, and Euxyl®.In certain embodiments, the preservative is an anti-oxidant. In otherembodiments, the preservative is a chelating agent.

Exemplary buffering agents include, but are not limited to, citratebuffer solutions, acetate buffer solutions, phosphate buffer solutions,ammonium chloride, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconicacid, calcium glycerophosphate, calcium lactate, propanoic acid, calciumlevulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid,tribasic calcium phosphate, calcium hydroxide phosphate, potassiumacetate, potassium chloride, potassium gluconate, potassium mixtures,dibasic potassium phosphate, monobasic potassium phosphate, potassiumphosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride,sodium citrate, sodium lactate, dibasic sodium phosphate, monobasicsodium phosphate, sodium phosphate mixtures, tromethamine, magnesiumhydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,isotonic saline, Ringer's solution, ethyl alcohol, etc., andcombinations thereof.

Exemplary lubricating agents include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, silica, talc, malt, glycerylbehenate, hydrogenated vegetable oils, polyethylene glycol, sodiumbenzoate, sodium acetate, sodium chloride, leucine, magnesium laurylsulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include, but are not limited to, almond, apricot kernel,avocado, babassu, bergamot, black current seed, borage, cade, camomile,canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, codliver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose,fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop,isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon,litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink,nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel,peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary,safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, sheabutter, silicone, soybean, sunflower, tea tree, thistle, tsubaki,vetiver, walnut, and wheat germ oils. Exemplary oils include, but arenot limited to, butyl stearate, caprylic triglyceride, caprictriglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,silicone oil, and combinations thereof.

Liquid dosage forms for oral and parenteral administration include, butare not limited to, pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active ingredients, the liquid dosage forms may comprise inertdiluents commonly used in the art such as, for example, water or othersolvents, solubilizing agents and emulsifiers such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, the oral compositions caninclude adjuvants such as wetting agents, emulsifying and suspendingagents, sweetening, flavoring, and perfuming agents. In certainembodiments for parenteral administration, vaccine nanocarriers of theinvention are mixed with solubilizing agents such as Cremophor®,alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins,polymers, and combinations thereof.

Injectable formulations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Asterile injectable preparation may be a sterile injectable solution,suspension or emulsion in a nontoxic parenterally acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedor dispersed in sterile water or other sterile injectable medium priorto use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform may be accomplished by dissolving or suspending the drug in an oilvehicle.

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing vaccine nanocarriers ofthis invention with suitable non-irritating excipients such as cocoabutter, polyethylene glycol or a suppository wax which are solid atambient temperature but liquid at body temperature and therefore melt inthe rectum or vaginal cavity and release active ingredient.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activeingredient is mixed with at least one inert, pharmaceutically acceptableexcipient such as sodium citrate or dicalcium phosphate and/or a)fillers or extenders such as starches, lactose, sucrose, glucose,mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may comprise buffering agents.

Solid compositions of a similar type may be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugar as well as high molecular weight polyethylene glycols and thelike. Solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. Solid compositions of asimilar type may be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

Active ingredients can be in micro-encapsulated form with one or moreexcipients as noted above. Solid dosage forms of tablets, dragees,capsules, pills, and granules can be prepared with coatings and shellssuch as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms, active ingredient may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms maycomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, dosage forms may comprise buffering agents.They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

Dosage forms for topical and/or transdermal administration of vaccinenanocarriers in accordance with the invention may include ointments,pastes, creams, lotions, gels, powders, solutions, sprays, inhalantsand/or patches. Generally, active ingredient is admixed under sterileconditions with a pharmaceutically acceptable excipient and/or anyneeded preservatives and/or buffers as may be required. Additionally,the present invention contemplates the use of transdermal patches, whichoften have the added advantage of providing controlled delivery of anactive ingredient to the body. Such dosage forms may be prepared, forexample, by dissolving and/or dispensing the active ingredient in theproper medium. Alternatively or additionally, the rate may be controlledby either providing a rate controlling membrane and/or by dispersing theactive ingredient in a polymer matrix and/or gel.

Suitable devices for use in delivering intradermal pharmaceuticalcompositions described herein include short needle devices such as thosedescribed in U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288;4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal compositionsmay be administered by devices which limit the effective penetrationlength of a needle into the skin, such as those described in PCTpublication WO 99/34850 and functional equivalents thereof. Jetinjection devices which deliver liquid vaccines to the dermis via aliquid jet injector and/or via a needle which pierces the stratumcorneum and produces a jet which reaches the dermis are suitable. Jetinjection devices are described, for example, in U.S. Pat. Nos.5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189;5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335;5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880;4,940,460; and PCT publications WO 97/37705 and WO 97/13537. Ballisticpowder/particle delivery devices which use compressed gas to acceleratevaccine in powder form through the outer layers of the skin to thedermis are suitable. Alternatively or additionally, conventionalsyringes may be used in the classical mantoux method of intradermaladministration.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,and/or sold in a formulation suitable for pulmonary administration viathe buccal cavity. Such a formulation may comprise dry particles whichcomprise the active ingredient and which have a diameter in the rangefrom about 0.5 μm to about 7 μm or from about 1 μm to about 6 μm. Suchcompositions are conveniently in the form of dry powders foradministration using a device comprising a dry powder reservoir to whicha stream of propellant may be directed to disperse the powder and/orusing a self propelling solvent/powder dispensing container such as adevice comprising the active ingredient dissolved and/or suspended in alow-boiling propellant in a sealed container. Such powders compriseparticles wherein at least 98% of the particles by weight have adiameter greater than 0.5 μm and at least 95% of the particles by numberhave a diameter less than 7 μm. Alternatively, at least 95% of theparticles by weight have a diameter greater than 1 μm and at least 90%of the particles by number have a diameter less than 6 μm. Dry powdercompositions may include a solid fine powder diluent such as sugar andare conveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50% to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1% to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic and/or solid anionic surfactant and/or a solid diluent(which may have a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may provide the active ingredient in the form of droplets of asolution and/or suspension. Such formulations may be prepared, packaged,and/or sold as aqueous and/or dilute alcoholic solutions and/orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization and/oratomization device. Such formulations may further comprise one or moreadditional ingredients including, but not limited to, a flavoring agentsuch as saccharin sodium, a volatile oil, a buffering agent, a surfaceactive agent, and/or a preservative such as methylhydroxybenzoate. Thedroplets provided by this route of administration may have an averagediameter in the range from about 0.1 μm to about 200 μm.

The formulations described herein as being useful for pulmonary deliveryare useful for intranasal delivery of a pharmaceutical composition ofthe invention. Another formulation suitable for intranasaladministration is a coarse powder comprising the active ingredient andhaving an average particle from about 0.2 μm to about 500 μm. Such aformulation is administered in the manner in which snuff is taken, i.e.by rapid inhalation through the nasal passage from a container of thepowder held close to the nares.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe active ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition of theinvention may be prepared, packaged, and/or sold in a formulationsuitable for buccal administration. Such formulations may, for example,be in the form of tablets and/or lozenges made using conventionalmethods, and may, for example, 0.1% to 20% (w/w) active ingredient, thebalance comprising an orally dissolvable and/or degradable compositionand, optionally, one or more of the additional ingredients describedherein. Alternately, formulations suitable for buccal administration maycomprise a powder and/or an aerosolized and/or atomized solution and/orsuspension comprising the active ingredient. Such powdered, aerosolized,and/or aerosolized formulations, when dispersed, may have an averageparticle and/or droplet size in the range from about 0.1 μm to about 200μm, and may further comprise one or more of the additional ingredientsdescribed herein.

A pharmaceutical composition of the invention may be prepared, packaged,and/or sold in a formulation suitable for ophthalmic administration.Such formulations may, for example, be in the form of eye dropsincluding, for example, a 0.1%/1.0% (w/w) solution and/or suspension ofthe active ingredient in an aqueous or oily liquid excipient. Such dropsmay further comprise buffering agents, salts, and/or one or more otherof the additional ingredients described herein. Otheropthalmically-administrable formulations which are useful include thosewhich comprise the active ingredient in microcrystalline form and/or ina liposomal preparation. Ear drops and/or eye drops are contemplated asbeing within the scope of this invention.

General considerations in the formulation and/or manufacture ofpharmaceutical agents may be found, for example, in Remington: TheScience and Practice of Pharmacy 21^(st) ed., Lippincott Williams &Wilkins, 2005.

Administration

In some embodiments, a therapeutically effective amount of an inventivevaccine nanocarrier composition is delivered to a patient and/or animalprior to, simultaneously with, and/or after diagnosis with a disease,disorder, and/or condition. In some embodiments, a therapeutic amount ofan inventive composition is delivered to a patient and/or animal priorto, simultaneously with, and/or after onset of symptoms of a disease,disorder, and/or condition. In some embodiments, the amount of a vaccinenanocarrier is sufficient to treat, alleviate, ameliorate, relieve,delay onset of, inhibit progression of, reduce severity of, and/orreduce incidence of one or more symptoms or features of the disease,disorder, and/or condition. In some embodiments, the amount of a vaccinenanocarrier is sufficient to elicit a detectable immune response in asubject. In some embodiments, the amount of a vaccine nanocarrier issufficient to elicit a detectable antibody response in a subject. Insome embodiments, the amount of a vaccine nanocarrier is sufficient toelicit a detectable T cell response in a subject. In some embodiments,the amount of a vaccine nanocarrier is sufficient to elicit a detectableantibody and T cell response in a subject. In some embodiments, anadvantage of the nanocarriers provided is that the nanocarriers canelicit potent responses with a much lower concentration of antigen thanrequired with a conventional vaccine.

The compositions, according to the method of the present invention, maybe administered using any amount and any route of administrationeffective for treatment. The exact amount required will vary fromsubject to subject, depending on the species, age, and general conditionof the subject, the severity of the infection, the particularcomposition, its mode of administration, its mode of activity, and thelike. The compositions of the invention are typically formulated indosage unit form for ease of administration and uniformity of dosage. Itwill be understood, however, that the total daily usage of thecompositions of the present invention will be decided by the attendingphysician within the scope of sound medical judgment. The specifictherapeutically effective dose level for any particular subject ororganism will depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; the activity of thespecific active ingredient employed; the specific composition employed;the age, body weight, general health, sex and diet of the subject; thetime of administration, route of administration, and rate of excretionof the specific active ingredient employed; the duration of thetreatment; drugs used in combination or coincidental with the specificactive ingredient employed; and like factors well known in the medicalarts.

The pharmaceutical compositions of the present invention may beadministered by any route. In some embodiments, the pharmaceuticalcompositions of the present invention are administered by a variety ofroutes, including oral, intravenous, intramuscular, intra-arterial,intramedullary, intrathecal, subcutaneous, intraventricular,transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical(as by powders, ointments, creams, and/or drops), transdermal, mucosal,nasal, buccal, enteral, sublingual; by intratracheal instillation,bronchial instillation, and/or inhalation; and/or as an oral spray,nasal spray, and/or aerosol. Specifically contemplated routes are oraladministration, intravenous injection, intramuscular injection, and/orsubcutaneous injection. In some embodiments, inventive vaccinenanocarriers are administered parenterally. In some embodiments,inventive vaccine nanocarriers are administered intravenously. In someembodiments, inventive vaccine nanocarriers are administered orally.

In general the most appropriate route of administration will depend upona variety of factors including the nature of the vaccine nanocarrier(e.g., its stability in the environment of the gastrointestinal tract),the condition of the subject (e.g., whether the subject is able totolerate oral administration), etc. The invention encompasses thedelivery of the inventive pharmaceutical composition by any appropriateroute taking into consideration likely advances in the sciences of drugdelivery.

In certain embodiments, the vaccine nanocarriers of the invention may beadministered in amounts ranging from about 0.001 mg/kg to about 100mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg toabout 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or fromabout 1 mg/kg to about 25 mg/kg, of subject body weight per day, one ormore times a day, to obtain the desired therapeutic effect. The desireddosage may be delivered three times a day, two times a day, once a day,every other day, every third day, every week, every two weeks, everythree weeks, or every four weeks. In certain embodiments, the desireddosage may be delivered using multiple administrations (e.g., two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, or more administrations).

In some embodiments, the present invention encompasses “therapeuticcocktails” comprising populations of inventive vaccine nanocarriers. Insome embodiments, all of the vaccine nanocarriers within a population ofvaccine nanocarriers comprise a single species of targeting moiety whichcan bind to multiple targets (e.g. can bind to both SCS-Mph and FDCs).In some embodiments, different vaccine nanocarriers within a populationof vaccine nanocarriers comprise different targeting moieties, and allof the different targeting moieties can bind to the same target. In someembodiments, different vaccine nanocarriers comprise different targetingmoieties, and all of the different targeting moieties can bind todifferent targets. In some embodiments, such different targets may beassociated with the same cell type. In some embodiments, such differenttargets may be associated with different cell types.

Where appropriate, the nanoparticle bioconjugates of the invention maybe administered per se (neat) or in the form of a pharmaceuticallyacceptable salts. When used in medicine the salts should bepharmaceutically acceptable, but non-pharmaceutically acceptable saltsmay conveniently be used to prepare pharmaceutically acceptable saltsthereof and are not excluded from the scope of the invention. Suchpharmaceutically acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulphuric,nitric, phosphoric, maleic, acetic, salicyclic, p-toluene sulphonic,tartaric, citric, methane sulphonic, formic, malonic, succinic,naphthalene-2-sulphonic, and benzene sulphonic. Also, pharmaceuticallyacceptable salts can be prepared as alkaline metal or alkaline earthsalts, such as sodium, potassium or calcium salts of the carboxylic acidgroup.

Encapsulating substances for the preparation of enteric-coated oralformulations include cellulose acetate phthalate, polyvinyl acetatephthalate, hydroxypropyl methylcellulose phthalate and methacrylic acidester copolymers. Solid oral formulations such as capsules or tabletsare preferred. Elixirs and syrups also are well known oral formulations.The components of aerosol formulations include solubilized activeingredients, antioxidants, solvent blends and propellants for solutionformulations, and micronized and suspended active ingredients,dispersing agents and propellants for suspension formulations. The oral,aerosol and nasal formulations of the invention can be distinguishedfrom injectable preparations of the prior art because such formulationsmay be nonaseptic, whereas injectable preparations must be aseptic.

In some embodiments of the invention, administration of the nanocarriersoccurs in a plurality of doses. For example, 2, 3, 4, 5, 6, 7, 8, 9, or10 doses may be administered to a patient. As-needed dosing is alsowithin the scope of the invention, and may include recurring doses on aregular schedule. The doses may be separated by any interval appropriateto achieve the desired biological effect, such as between 1 day and 1year, or between 1 day and 1 month, or between 1 day and 1 week.

In some embodiments, the first dose of nanocarriers elicits T cellproliferation in the subject receiving the dose. In some embodiments,after administration of a first dose of nanocarriers to a patient, theserum concentration of T cells in the subject is greater than 200 ng/ml,or greater than 250 ng/ml, or greater than 300 ng/ml, or greater than400 ng/ml, or greater than 500 ng/ml. In some embodiments, such serumconcentrations of T cells in the subject is achieved and/or maintainedafter administration of a subsequent dose of the nanocarriers.

In some embodiments of the invention, administering of nicotinenanoparticles is carried out with a subject having a baseline IgGanti-nicotine antibody serum concentration of less than 100 ng/ml, orless than 50 ng/ml, or less than 25 ng/ml. In preferred suchembodiments, post-administration anti-nicotine IgG antibody peak serumconcentration in the subject is more than 200 ng/ml, or more than 300ng/ml, or more than 500 ng/ml.

In some embodiments, nicotine nanoparticles according to the inventionare administered to a subject that has not been previously vaccinatedagainst nicotine. In preferred such embodiments, a post-administrationanti-nicotine antibody peak concentration is observed in the subjectthat is greater than or equal to 100 ng/ml, or greater than 200 ng/ml,or greater than 500 ng/ml.

Combination Therapies

It will be appreciated that vaccine nanocarriers and pharmaceuticalcompositions of the present invention can be employed in combinationtherapies. The particular combination of therapies (therapeutics orprocedures) to employ in a combination regimen will take into accountcompatibility of the desired therapeutics and/or procedures and thedesired therapeutic effect to be achieved. It will be appreciated thatthe therapies employed may achieve a desired effect for the same purpose(for example, an inventive vaccine nanocarrier useful for vaccinatingagainst a particular type of microbial infection may be administeredconcurrently with another agent useful for treating the same microbialinfection), or they may achieve different effects (e.g., control of anyadverse effects attributed to the vaccine nanocarrier).

In some embodiments, pharmaceutical compositions of the presentinvention may be administered either alone or in combination with one ormore other therapeutic agents. By “in combination with,” it is notintended to imply that the agents must be administered at the same timeand/or formulated for delivery together, although these methods ofdelivery are within the scope of the invention. The compositions can beadministered concurrently with, prior to, or subsequent to, one or moreother desired therapeutics or medical procedures. In general, each agentwill be administered at a dose and/or on a time schedule determined forthat agent. Additionally, the invention encompasses the delivery of theinventive pharmaceutical compositions in combination with agents thatmay improve their bioavailability, reduce and/or modify theirmetabolism, inhibit their excretion, and/or modify their distributionwithin the body.

The particular combination of therapies (therapeutics and/or procedures)to employ in a combination regimen will take into account compatibilityof the desired therapeutics and/or procedures and/or the desiredtherapeutic effect to be achieved. It will be appreciated that thetherapies employed may achieve a desired effect for the same disorder(for example, an inventive vaccine nanocarrier may be administeredconcurrently with another therapeutic agent used to treat the samedisorder), and/or they may achieve different effects (e.g., control ofany adverse effects attributed to the vaccine nanocarrier). In someembodiments, vaccine nanocarriers of the invention are administered witha second therapeutic agent that is approved by the U.S. Food and DrugAdministration.

In will further be appreciated that therapeutically active agentsutilized in combination may be administered together in a singlecomposition or administered separately in different compositions.

In general, it is expected that agents utilized in combination with beutilized at levels that do not exceed the levels at which they areutilized individually. In some embodiments, the levels utilized incombination will be lower than those utilized individually.

In some embodiments, inventive vaccine nanocarriers may be administeredin combination with an agent, including, for example, therapeutic,diagnostic, and/or prophylactic agents. Exemplary agents to be deliveredin accordance with the present invention include, but are not limitedto, small molecules, organometallic compounds, nucleic acids, proteins(including multimeric proteins, protein complexes, etc.), peptides,lipids, carbohydrates, hormones, metals, radioactive elements andcompounds, drugs, vaccines, immunological agents, etc., and/orcombinations thereof.

In certain embodiments, vaccine nanocarriers which delay the onsetand/or progression of a particular microbial infection may beadministered in combination with one or more additional therapeuticagents which treat the symptoms of microbial infection. To give but oneexample, upon exposure to rabies virus, nanocarriers comprisingimmunomodulatory agents useful for vaccination against rabies virus maybe administered in combination with one or more therapeutic agentsuseful for treatment of symptoms of rabies virus (e.g. antipsychoticagents useful for treatment of paranoia that is symptomatic of rabiesvirus infection).

In some embodiments, pharmaceutical compositions comprising inventivevaccine nanocarriers comprise less than 50% by weight, less than 40% byweight, less than 30% by weight, less than 20% by weight, less than 15%by weight, less than 10% by weight, less than 5% by weight, less than 1%by weight, or less than 0.5% by weight of an agent to be delivered.

The pharmaceutical compositions provided herein may further comprise oneor more additional biologically active substances. Generally,non-limiting examples of such substances include eukaryotic andprokaryotic cells, viruses, vectors, proteins, peptides, nucleic acids,polysaccharides and carbohydrates, lipids, glycoproteins, and syntheticorganic and inorganic drugs that exert a biological effect whenadministered to an animal. Combinations of such biologically activesubstances are also within the scope of the invention. For ease ofreference, and unless indicated otherwise, the term “active substance”is also used to include detectable compounds such as radiopaquecompounds including air and barium, magnetic compounds, and the like.The additional active substances can be soluble or insoluble in water.Further examples of biologically active substances includeantiangiogenesis factors, antibodies, antimicrobials, antimalarials,amebicides, antiprotazoal, antifungals, antivirals, antineoplasticcompounds, growth factors hormones, enzymes, immunoactives, and drugssuch as steroids or antibiotics. Non-limiting examples of these andother classes of biologically active substances are listed below and maybe encapsulated in the nanoparticle delivery system for the purposes ofdelivery to targeted cells or tissues.

In some embodiments, vaccine nanocarriers are administered incombination with one or more small molecules and/or organic compoundswith pharmaceutical activity. In some embodiments, the agent is aclinically-used drug. In some embodiments, the drug is an anti-canceragent, antibiotic, anti-viral agent, anti-HIV agent, anti-parasiteagent, anti-protozoal agent, anesthetic, anticoagulant, inhibitor of anenzyme, steroidal agent, steroidal or non-steroidal anti-inflammatoryagent, antihistamine, immunosuppressant agent, anti-neoplastic agent,antigen, vaccine, antibody, decongestant, sedative, opioid, analgesic,anti-pyretic, birth control agent, hormone, prostaglandin,progestational agent, anti-glaucoma agent, ophthalmic agent,anti-cholinergic, analgesic, anti-depressant, anti-psychotic,neurotoxin, hypnotic, tranquilizer, anti-convulsant, muscle relaxant,anti-Parkinson agent, anti-spasmodic, muscle contractant, channelblocker, miotic agent, anti-secretory agent, anti-thrombotic agent,anticoagulant, anti-cholinergic, β-adrenergic blocking agent, diuretic,cardiovascular active agent, vasoactive agent, vasodilating agent,anti-hypertensive agent, angiogenic agent, modulators ofcell-extracellular matrix interactions (e.g. cell growth inhibitors andanti-adhesion molecules), inhibitors of DNA, RNA, or protein synthesis,etc.

In certain embodiments, a small molecule agent can be any drug. In someembodiments, the drug is one that has already been deemed safe andeffective for use in humans or animals by the appropriate governmentalagency or regulatory body. For example, drugs approved for human use arelisted by the FDA under 21 C.F.R. §§330.5, 331 through 361, and 440through 460, incorporated herein by reference; drugs for veterinary useare listed by the FDA under 21 C.F.R. §§500 through 589, incorporatedherein by reference. All listed drugs are considered acceptable for usein accordance with the present invention.

A more complete listing of classes and specific drugs suitable for usein the present invention may be found in Pharmaceutical Drugs:Syntheses, Patents, Applications by Axel Kleemann and Jurgen Engel,Thieme Medical Publishing, 1999, Goodman and Gilman, The PharmacologicalBasis of Therapeutics. 9th ed. McGraw-Hill 1996, and the Merck Index: AnEncyclopedia of Chemicals, Drugs and Biologicals, Ed. by Budavari etal., CRC Press, 1996. The relevant portions of these texts areincorporated herein by reference.

In some embodiments, vaccine nanocarriers are administered incombination with one or more nucleic acids (e.g. functional RNAs,functional DNAs, etc.) to a specific location such as a tissue, cell, orsubcellular locale. For example, inventive vaccine nanocarriers whichare used to delay the onset and/or progression of a particular microbialinfection may be administered in combination with RNAi agents whichreduce expression of microbial proteins. Molecular properties of nucleicacids are described in the section above entitled “Nucleic AcidTargeting Moieties.”

In some embodiments, vaccine nanocarriers are administered incombination with one or more proteins or peptides. In some embodiments,the agent to be delivered may be a peptide, hormone, erythropoietin,insulin, cytokine, antigen for vaccination, etc. In some embodiments,the agent to be delivered may be an antibody and/or characteristicportion thereof. Molecular properties of which are described in thesection above entitled “Protein Targeting Moieties.”

In some embodiments, vaccine nanocarriers are administered incombination with one or more carbohydrates, such as a carbohydrate thatis associated with a protein (e.g. glycoprotein, proteogycan, etc.). Acarbohydrate may be natural or synthetic. A carbohydrate may also be aderivatized natural carbohydrate. In certain embodiments, a carbohydratemay be a simple or complex sugar. In certain embodiments, a carbohydrateis a monosaccharide, including but not limited to glucose, fructose,galactose, and ribose. In certain embodiments, a carbohydrate is adisaccharide, including but not limited to lactose, sucrose, maltose,trehalose, and cellobiose. In certain embodiments, a carbohydrate is apolysaccharide, including but not limited to cellulose, microcrystallinecellulose, hydroxypropyl methylcellulose (HPMC), methylcellulose (MC),dextrose, dextran, glycogen, xanthan gum, gellan gum, starch, andpullulan. In certain embodiments, a carbohydrate is a sugar alcohol,including but not limited to mannitol, sorbitol, xylitol, erythritol,malitol, and lactitol. Molecular properties of carbohydrates aredescribed in the section above entitled “Vaccine Nanocarriers ComprisingCarbohydrates.”

In some embodiments, vaccine nanocarriers are administered incombination with one or more lipids, such as a lipid that is associatedwith a protein (e.g. lipoprotein). Exemplary lipids that may be used inaccordance with the present invention include, but are not limited to,oils, fatty acids, saturated fatty acid, unsaturated fatty acids,essential fatty acids, cis fatty acids, trans fatty acids, glycerides,monoglycerides, diglycerides, triglycerides, hormones, steroids (e.g.,cholesterol, bile acids), vitamins (e.g. vitamin E), phospholipids,sphingolipids, and lipoproteins. Molecular properties of lipids aredescribed in the section above entitled “Lipid Vaccine Nanocarriers.”

Further examples of therapeutic agents include the following:

Antineoplastic compounds. Examples include nitrosoureas (e.g.,carmustine, lomustine, semustine, strepzotocin), methylhydrazines (e.g.,procarbazine, dacarbazine).

Steroid hormones. Examples include glucocorticoids, estrogens,progestins, androgens, tetrahydrodesoxycaricosterone, cytokines andgrowth factors, Asparaginase.

Immunoactive compounds. Examples include immunosuppressives (e.g.,pyrimethamine, trimethopterin, penicillamine, cyclosporine,azathioprine) and immunostimulants (e.g., levamisole, diethyldithiocarbamate, enkephalins, endorphins).

Antimicrobial compounds. Examples include antibiotics, e.g., β-lactam,penicillin, cephalosporins, carbapenims and monobactams, β-lactamaseinhibitors, aminoglycosides, macrolides, tetracyclins, andspectinomycin.

Antimalarials, amebicides, antiprotazoal, antivirals, and antifungals.Examples include amphotericin β, acyclovir, idoxuridine, ribavirin,trifluridine, vidarbine, gancyclovir. Further antiviral agents includereverse transcriptase inhibitors and nucleoside analogs, e.g. ddI, ddC,3TC, ddA, AZT; protease inhibitors, e.g., Invirase, ABT-538; inhibitorsof in RNA processing, e.g., ribavirin.

Parasiticides, antihalmintics, radiopharmaceutics, gastrointestinaldrugs, hematologic compounds, and immunoglobulins. Blood clottingproteins, e.g., antihemophilic factor and factor IX complex.Anticoagulants, e.g., dicumarol, and heparin Na. Fibrolysin inhibitors,such as tranexamic acid.

Cardiovascular Drugs and peripheral antiadrenergic drugs. Centrallyacting antihypertensive drugs, e.g., methyldopa, methyldopa HCl,Antihypertensive direct vasodilators, e.g., diazoxide, hydralazine HCl.Drugs affecting renin-angiotensin system. Peripheral vasodilators suchas phentolamine. Antianginal drugs and cardiac glycosides. Inodilators,e.g., aminone, milrinone, enoximone, fenoximone, imazodan, andsulmazole. Antidysrhythmics, calcium entry blockers, and drugs affectingblood lipids, e.g., ranitidine, bosentan, rezulin.

Respiratory Drugs. Sypathomimetic drugs, e.g., albuterol, bitolterolmesylate, dobutamine HCl, dopamine HCl, ephedrine So, epinephrine,fenfluramine HCl, isoproterenol HCl, methoxamine HCl, norepinephrinebitartrate, phenylephrine HCl, ritodrine HCl.

Cholinomimetic drugs, e.g., acetylcholine Cl. Anticholinesterases, e.g.,edrophonium Cl. Cholinesterase reactivators. Adrenergic blocking drugs,e.g., acebutolol HCl, atenolol, esmolol HCl, labetalol HCl, metoprolol,nadolol, phentolamine mesylate, and propanolol HCl. Antimuscarinicdrugs, e.g., anisotropine methylbromide, atropine S04, clinidium Br,glycopyrrolate, ipratropiur Br, and scopolamine HBr.

Neurotransmitters and neurotransmitter agents, e.g., acetylcholine,adenosine, adenosine triphosphate. Amino acid neurotransmitters, e.g.,excitatory amino acids, GABA, glycine. Biogenic amine neurotransmitters,e.g., dopamine, epinephrine, histamine, norepinephrine, octopamine,serotonin, tyramine Neuropeptides, nitric oxide, and K+ channel toxins.Antiparkinson drugs. Examples include amaltidine HCl, benztropinemesylate, e.g., carbidopa.

Hormones. Pituitary hormones, e.g., chorionic gonadotropin, cosyntropin,menotropins, somatotropin, iorticotropin, protirelin, thyrotropin,vasopressin, and lypressin. Adrenal hormones, e.g., beclomethasonedipropionate, betamethasone, dexamethasone, and triamcinolone.Pancreatic hormones, e.g., glucagon, and insulin. Parathyroid hormone,e.g., dihydrochysterol. Thyroid hormones, e.g., calcitonin etidronatedisodium, levothyroxine Na, liothyronine Na, liotrix, thyroglobulin,teriparatide acetate. Antithyroid drugs. Estrogenic hormones. Progestinsand antagonists, and hormonal contraceptives. Testicular hormones.Gastrointestinal hormones, e.g., cholecystokinin, enteroglycan, galanin,gastric inhibitory polypeptide, epidermal growth factor-urogastrone,gastric inhibitory polypeptide, gastrin-releasing peptide, gastrins,pentagastrin, tetragastrin, motilin, peptide YY, secretin, vasoactiveintestinal peptide, sincalide.

Enzymes. Examples include hyaluronidase, streptokinase, tissueplasminogen activator, urokinase, PGE-adenosine deaminase.

Peptides and proteins. The nanoparticle bioconjugate system mayencapsulate peptides/proteins and/or may have surfaces functionalizedwith peptides/proteins. Examples of peptides and proteins includeankyrins, arrestins, bacterial membrane proteins, clathrin, connexins,ovalbumin, viral proteins from capsid or the envelop like HA or VSVG,dystrophin, endothelin receptor, spectrin, selectin, cytokines;chemokines; growth factors, insulin, erythropoietin (EPO), tumornecrosis factor (TNF), neuropeptides, neuropeptide Y, neurotensin,transforming growth factor α, transforming growth factor β, interferon(IFN), and hormones, growth inhibitors, e.g., genistein, steroids etc;glycoproteins, e.g., ABC transporters, platelet glycoproteins, GPIb-IXcomplex, GPIIb-IIIa complex, vitronectin, thrombomodulin, CD4, CD55,CD58, CD59, CD44, lymphocye function-associated antigen, intercellularadhesion molecule, vascular cell adhesion molecule, Thy-1, antiporters,CA-15-3 antigen, fibronectins, laminin, myelin-associated glycoprotein,GAP, GAP-43.

Cytokines and Cytokine Receptors. Examples of cytokines and receptorsthereof which may be delivered via the nanoparticle carriers of thepresent invention include, but are not limited to: Interleukin-1 (IL-1),IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL11, IL-12,IL-13, IL-14, L-15, IL-16, IL-17, IL-18, IL-1 receptor, IL-2 receptor,IL-3 receptor, IL-4 receptor, IL-5 receptor, L-6 receptor, L-7 receptor,IL-8 receptor, IL-9 receptor, IL-10 receptor, IL-11 receptor, IL-12receptor, IL-13 receptor, L-14 receptor, IL-15 receptor, IL-16 receptor,IL-17 receptor, IL-18 receptor, lymphokine inhibitory factor, macrophagecolony stimulating factor, platelet derived growth factor, stem cellfactor, tumor growth factor β, tumor necrosis factor, lymphotoxin, Fas,granulocyte colony stimulating factor, granulocyte macrophage colonystimulating factor, interferon α, interferon β, interferon γ.

Growth Factors, Protein Hormones, and receptors thereof. Examplesinclude erythropoietin, angiogenin, hepatocyte growth factor, fibroblastgrowth factor, keratinocyte growth factor, nerve growth factor, tumorgrowth factor α, thrombopoietin, thyroid stimulating factor, thyroidreleasing hormone, neurotrophin, epidermal growth factor, VEGF, ciliaryneurotrophic factor, LDL, somatomedin, insulin growth factor,insulin-like growth factor I and II.

Chemokines and receptors thereof. Examples include: ENA-78, ELC, GRO-α,GRO-β, GRO-γ, HRG, LIF, IP-10, MCP-1, MCP-2, MCP-3, MCP-4, MIP-1α,MIP-1β, MIG, MDC, NT-3, NT-4, SCF, LIF, leptin, RANTES, lymphotactin,eotaxin-1, eotaxin-2, TARC, TECK, WAP-1, WAP-2, GCP-1, GCP-2,α-chemokine receptors: CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6,CXCR713-chemokine receptors: CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7.

Antibodies. Examples of antibodies which may be used as targeting ligandvia the nanoparticle carriers of the present invention include: (a)anti-cluster of differentiation antigen CD-1 through CD-166 and theligands or counter receptors for these molecules; (b) anti-cytokineantibodies, e.g., anti-IL-1 through anti-IL-18 and the receptors forthese molecules; (c) anti-immune receptor antibodies, antibodies againstT cell receptors, major histocompatibility complexes I and II, B cellreceptors, selectin killer inhibitory receptors, killer activatingreceptors, OX-40, MadCAM-1, Gly-CAM1, integrins, cadherens,sialoadherens, Fas, CTLA-4, Fcy-receptors, Fcα-receptors, Fcε-receptors,Fcμt-receptors, and their ligands; (d) anti-metalloproteinaseantibodies, e.g., collagenase, MMP-1 through MMP-8, TIMP-1, TIMP-2;anti-cell lysislproinflammatory molecules, e.g., perforin, complementcomponents, prostanoids, nitron oxide, thromboxanes; and (e)anti-adhesion molecules, e.g., carcioembryonic antigens, lamins,fibronectins.

Specific examples of known therapeutics which may be delivered via ananoparticle delivery of the invention include, but are not limited to:(a) Capoten, Monopril, Pravachol, Avapro, Plavix, Cefzil,Duricef/Ultracef, Azactam, Videx, Zerit, Maxipime, VePesid, Paraplatin,Platinol, Taxol, UFT, Buspar, Serzone, Stadol NS, Estrace, Glucophage(Bristol-Myers Squibb); (b) Ceclor, Lorabid, Dynabac, Prozac, Darvon,Permax, Zyprexa, Humalog, Axid, Gemzar, Evista (Eli Lily); (c)VasotecNaseretic, Mevacor, Zocor, Prinivil/Prinizide, Plendil,CozaarIHyzaar, Pepcid, Prilosec, Primaxin, Noroxin, Recombivax HB,Varivax, TimopticIXE, Trusopt, Proscar, Fosamax, Sinemet, Crixivan,Propecia, Vioxx, Singulair, Maxalt, Ivermectin (Merck & Co.); (d)Diflucan, Unasyn, Sulperazon, Zithromax, Trovan, Procardia XL, Cardura,Norvasc, Dofetilide, Feldene, Zoloft, Zeldox, Glucotrol XL, Zyrtec,Eletriptan, Viagra, Droloxifene, Aricept, Lipitor (Pfizer); (e) Vantin,Rescriptor, Vistide, Genotropin, Micronase/Glyn./Glyb., Fragrnin, TotalMedrol, Xanaxlalprazolam, Sermion, Halcionitriazolam, Freedox, Dostinex,Edronax, Mirapex, Pharmorubicin, Adriamycin, Carnptosar, Remisar,Depo-Provera, Caverject, Detrusitol, Estring, Healon, Xalatan, Rogaine(Pharmacia & Upjohn); (f) Lopid, Accrupil, Dilantin, Cognex, Neurontin,Loestrin, Dilzem, Fempatch, Estrostep, Rezulin, Lipitor, Omnicef,FemHRT, Suramin, and Clinafloxacin (Warner Lambert).

Those skilled in the art will recognize that this is an exemplary, notcomprehensive, list of therapeutic, diagnostic, and/or prophylacticagents that can be delivered in combination with the vaccinenanocarriers of the present invention. Any therapeutic, diagnostic,and/or prophylactic agent may be administered with vaccine nanocarriersin accordance with the present invention.

Kits

The invention provides a variety of kits comprising one or more of thenanocarriers of the invention. For example, the invention provides a kitcomprising an inventive vaccine nanocarrier and instructions for use. Akit may comprise multiple different vaccine nanocarriers. A kit maycomprise any of a number of additional components or reagents in anycombination. All of the various combinations are not set forthexplicitly but each combination is included in the scope of theinvention.

According to certain embodiments of the invention, a kit may include,for example, (i) a vaccine nanocarrier comprising at least oneimmunomodulatory agent, wherein the at least one immunomodulatory agentis capable of stimulating both a T cell and B cell response; (ii)instructions for administering the vaccine nanocarrier to a subject inneed thereof.

In certain embodiments, a kit may include, for example, (i) a vaccinenanocarrier comprising at least one immunomodulatory agent, wherein theat least one immunomodulatory agent is capable of stimulating both a Tcell and B cell response, at least one targeting moiety, and/or at leastone immunomodulatory agent; (ii) instructions for administering thevaccine nanocarrier to a subject in need thereof.

In certain embodiments, a kit may include, for example, (i) at least oneimmunomodulatory agent, wherein the at least one immunomodulatory agentis capable of stimulating both a T cell and B cell response; (ii) atleast one targeting moiety; (iii) at least one immunostimulatory agent;(iv) a polymeric matrix precursor; (v) lipids and amphiphilic entities;(vi) instructions for assembling inventive vaccine nanocarriers fromindividual components (i)-(v).

In some embodiments, the kit comprises an inventive nanocarrier andinstructions for mixing. Such kits, in some embodiments, also include animmunostimulatory agent and/or an immunomodulatory agent (e.g., a B cellor T cell antigen) The nanocarrier of such kits may comprise animmunomodulatory agent (e.g., a T cell antigen, such as a universal Tcell antigen) and/or a targeting moiety. The T cell antigen and/or thetargeting moiety may be on the surface of the nanocarrier. In someembodiments, the immunomodulatory agent and the antigen are the same. Insome embodiments, they are different.

Kits typically include instructions for use of inventive vaccinenanocarriers. Instructions may, for example, comprise protocols and/ordescribe conditions for production of vaccine nanocarriers,administration of vaccine nanocarriers to a subject in need thereof,etc. Kits generally include one or more vessels or containers so thatsome or all of the individual components and reagents may be separatelyhoused. Kits may also include a means for enclosing individualcontainers in relatively close confinement for commercial sale, e.g., aplastic box, in which instructions, packaging materials such asstyrofoam, etc., may be enclosed. An identifier, e.g., a bar code, radiofrequency identification (ID) tag, etc., may be present in or on the kitor in or one or more of the vessels or containers included in the kit.An identifier can be used, e.g., to uniquely identify the kit forpurposes of quality control, inventory control, tracking, movementbetween workstations, etc.

EXEMPLIFICATION Example 1 Subcapsular Sinus Macrophages in Lymph NodesClear Lymph-Borne Viruses and Present them to Antiviral B CellsMaterials and Methods

Method Summary

VSV-IND and VSV-NJ virions were purified from culture supernatants ofinfected BSRT7 cells and used either unmodified or fluorescently labeledwith Alexa-568 (red) or Alexa-488 (green). Fluorescent viruses used fortissue imaging were UV-irradiated to prevent generation ofnon-fluorescent progeny. Fluorescent labeling or UV-irradiation ofVSV-IND particles did not affect their antigenicity or their ability toelicit a calcium flux in VI10YEN cells (not shown). Followingfluorescent virus injection into footpads, draining popliteal LNs wereharvested for analysis by electron microscopy or to generate frozensections for immunostaining and confocal microscopy. To image adoptivelytransferred B cells in LNs, VI10YEN and wildtype B cells werefluorescently labeled and co-transferred by i.v. injection into wildtypeor mutant recipient mice. 18 hours later, when B cells had homed to Bcell follicles, mice were injected with labeled or unlabeled VSV in theright footpad. At different time intervals thereafter, the drainingpopliteal LN was observed by MP-IVM or harvested for confocal microscopyor for flow cytometry to analyze the activation state of virus-specificand control B cells. In some experiments, macrophages in the poplitealLN were depleted by sc injections of CLL, and animals were used forexperiments 7-10 days later. MP-IVM, electron microscopy,immunohistochemistry and flow cytometry for various markers wasperformed on LNs with and without prior CLL treatment. VSV propagationfrom the footpad injection site to the blood and other organs wasassessed by injecting a defined amount of live VSV into footpadsfollowed by tissue harvest at two hours or six hours after VSVinjection. To measure viral titers, tissues were homogenized and used inplaque assays. Some viral propagation experiments were performed aftercannulation of the thoracic duct.

Mice and Antibodies

C57BL/6 and BALB/c mice were purchased from Taconic Farms (Germantown,N.Y.). VI10YEN (Hangartner et al., 2003, Proc. Natl. Acad. Sci., USA,100:12883; incorporated herein by reference), C3^(−/−) (Wessels et al.,1995, Proc. Natl. Acad. Sci., USA, 92:11490; incorporated herein byreference), MHCII-EGFP (Boes et al., 2002, Nature, 418: 983;incorporated herein by reference), Act-EGFP (Wright et al., 2001, Blood,97:2278), and DH-LMP2A mice (Casola et al., 2004, Nat. Immunol., 5:317;incorporated herein by reference) were bred in barrier animal facilitiesat Harvard Medical School and the Immune Disease Institute (IDI).Radiation chimeras were generated by irradiation of Act(EGFP) mice withtwo doses of 650 rad and reconstitution with C57BL/6 bone marrow, andwere allowed to reconstitute for 8 weeks prior to use. In someexperiments, SCS macrophages were depleted by footpad injections of 30μl clodronate liposomes (CLL), 7-10 days prior to the experiment.

Clodronate was a gift of Roche Diagnostics GmbH, Mannheim, Germany.Other reagents for preparation of liposomes were: Phosphatidylcholine(LIPOID E PC, Lipoid GmbH, Ludwigshafen, Germany) and cholesterol(Sigma-Aldrich).

Mice were housed under specific pathogen-free and anti-viralantibody-free conditions in accordance with National Institutes ofHealth guidelines. All experimental animal procedures were approved bythe Institutional Animal Committees of Harvard Medical School and theIDI.

Antibodies were purchased from BD Biosciences (San Jose, Calif.), exceptanti-B220-Alexa647 (Invitrogen-Caltag), anti-LYVE-1 (Millipore-Upstate),goat-anti-rabbit-APC (Invitrogen), goat-anti-GFP-FITC (Rockland),anti-FITC-Alexa488 (Invitrogen), and Fab anti-IgM-FITC (JacksonImmunoresearch). The following antibodies were purchased fromAbD-Serotec: anti-CD68-Alexa647, anti-CD11b-Alexa647, F4/80-Alexa647,anti-CD169-FITC (3D6). The anti-idiotypic antibody 35.61 for detectionof the VI10 BCR in VI10YEN mice (Hangartner et al., 2003, Proc. Natl.Acad. Sci., USA, 100:12883; incorporated herein by reference) wasproduced from hybridoma supernatants according to standard methods.

Flow Cytometry

Flow cytometric analysis of blood samples was performed afterretro-orbital phlebotomy of mice and lysis of erythrocytes with ACKbuffer (0.15 M NH₄Cl, 1 mM KHCO₃, 0.1 mM EDTA (disodium salt), pH 7.2).Single-cell suspensions of LNs and spleens for flow cytometry weregenerated by careful mincing of tissues and subsequent digestion at 37°C. for 40 minutes in DMEM (Invitrogen-Gibco) in the presence of 250μg/ml liberase CI (Roche) plus 50 μg/ml DNase-I (Roche). After 20minutes of digestion, samples were vigorously passed through an 18 Gneedle to ensure complete organ dissociation. All flow cytometricanalyses were performed in FACS buffer containing PBS with 2 mM EDTA and2% FBS (Invitrogen-GIBCO) on a FACScalibur (BD Pharmingen), and analyzedby FlowJo software (Treestar Inc., Ashland, Oreg.). For calcium flux,cells were labeled with 4 μM Fluo-LOJO (Teflabs) in DMEM containing 10%FCS for 90 minutes at 37° C. Cells were spun through FCS and usedimmediately.

Viruses and VSV Plaque Assay

VSV serotypes Indiana (VSV-IND, Mudd-Summers derived clone, in vitrorescued (Whelan et al., 1995, Proc. Natl. Acad. Sci., USA, 92:8388;incorporated herein by reference) and plaque purified) or New Jersey(VSV-NJ, Pringle Isolate, plaque purified) were propagated at a MOI of0.01 on BSRT7 cells. Supernatants of infected cells were cleared fromcell debris by centrifugation at 2000×g, filtered through 0.45 μmsterile filters and subjected to ultracentrifugation at 40,000×g for 90minutes. Pellets were resuspended in PBS and purified byultracentrifugation (157,000×g, 60 minutes) through a cushion of 10%sucrose in NTE (0.5 mM NaCl, 10 mM Tris-HCl pH 7.5, 5 mM EDTA pH 8).After resuspension in PBS overnight, virus protein was quantified by BCAassay (Pierce), and infectivity was quantified by plaque assay. Somebatches were labeled with carboxylic acid succinimidyl esters ofAlexaFluor-488 or AlexaFluor-568 (Invitrogen-Molecular Probes) at a104-105-fold molar excess of Alexa dye over virus particles.Unconjugated dye was removed by ultracentrifugation through 10% sucrosein NTE, pellets resuspended in PBS and stored frozen. Infectivity of VSVpreparations was quantified by plaque assay on green monkey kidney cells(Vero). VSV titers from organs of infected mice were determinedsimilarly, after homogenization of the organs with a Potter-Elvejhemhomogenizer. When necessary, during viral preparation, the approximately4 ml supernatants from the 157,000×g ultracentrifugation were collectedand concentrated with a 10,000 MWCO Amicon Ultra (Millipore). In orderto account for residual infectivity in concentrated supernatants, VSVstocks were diluted to levels of infectivity equal to that of theconcentrated supernatants and calcium flux in VI10YEN B cells wascompared over further 100 fold dilutions of VSV and supernatant.UV-inactivated, AlexaFluor-568 labeled Adenovirus 5 (AdV5) was generatedfollowing standard procedures (Leopold et al., 1998, Human Gene Therapy,9:367; incorporated herein by reference). All infectious work wasperformed in designated BL2+ workspaces, in accordance withinstitutional guidelines, and approved by the Harvard Committee onMicrobiological Safety.

VSV Neutralization Assay

Serum of immunized mice was prediluted 40-fold in MEM containing 2% FCS.Serial two-fold dilutions were mixed with equal volumes of VSV (500pfu/ml) and incubated for 90 minutes at 37° C. in 5% CO₂. 100 μl ofserum-virus mixture was transferred onto Vero cell monolayers in 96-wellplates and incubated for 1 hour at 37° C. The monolayers were overlaidwith 100 μl DMEM containing 1% methylcellulose and incubated for 24hours at 37° C. Subsequently, the overlay was discarded, and themonolayer was fixed and stained with 0.5% crystal violet. The highestdilution of serum that reduced the number of plaques by 50% was taken astiter. To determine IgG titers, undiluted serum was pretreated with anequal volume of 0.1 mM β-mercaptoethanol in saline.

Adhesion Assays

96-well plates (Corning) were coated overnight with dilutions ofrecombinant murine VCAM-1-Fc or ICAM-1-Fc (R&D systems), or purifiedVSV-IND in PBS in triplicates. Negative control wells were coated with4% BSA, positive control wells were coated with 1 mg/ml poly-L-lysine.Plates were blocked for 1-2 h at 4° C. with Hanks Balanced Salt Solution(HBSS)/1% BSA and washed. Naïve B cells from VI10YEN or C57BL/6 micewere negatively selected by magnetic cell separation using CD43 magneticbeads (Miltenyi, Bergisch Gladbach, Germany) and added to the plates at3×10⁵/well in HBSS with 1% BSA, 1 mM Ca₂ ⁺ and 1 mM Mg₂ ⁺ in thepresence or absence of UV-inactivated VSV-IND (MOI of 1000) for 30minutes at 37° C. After gentle washing (3 times in HBSS with 1% BSA),plates were fixed for 10 minutes with PBS/10% glutaraldehyde, stainedfor 45 minutes with 0.5% crystal violet/20% methanol, and washed inwater. Dye was eluted by addition of 1% SDS and absorbance at 570 nm wasspectrophotometrically determined (SpectraMax340PC microplate reader andSoftmaxPro 3.1.2 software, Molecular Devices Corporation) after 30minutes.

Confocal Microscopy

For some analyses, C57BL/6 mice were injected into both hind footpadswith 20 AlexaFluor-568 or AlexaFluor-488 labeled VSV-IND or VSV-NJ for30 minutes. For other experiments, mice were transfused with 1×10⁷negatively selected naïve B cells from VI10YEN x MHCII-EGFP mice one dayprior to the experiment. At predetermined time points, popliteal LNswere fixed in situ by footpad injections of phosphate buffered L-lysinewith 1% paraformaldehyde/periodate (PLP). After removal of popliteal LNsand 3-5 hours incubation in PLP at 4° C., popliteal LNs were washed in0.1 M PBS, pH 7.2 and cryoprotected by an ascending series of 10%, 20%,and 30% sucrose in PBS. Samples were snap-frozen in TBS tissue freezingliquid (Triangle Biomedical Sciences, Durham N.C.) and stored at −80° C.Sections of 40 μm thickness were mounted on Superfrost Plus slides(Fisherbrand) and stained with fluorescent antibodies in a humidifiedchamber after Fc receptor blockade with 1 μg/ml antibody 2.4G2 (BDPharmingen). Samples were mounted in Fluor Save reagent solution(EMD-Calbiochem) and stored at 4° C. until analysis. Images werecollected with a BioRad confocal microscopy system using an OlympusBX50WI microscope and 10×/0.4 or 60×/1.2 W objectives. Images wereanalyzed using LaserSharp2000 software (BioRad Cell Science, HemelHempstead, Great Britain) and Photoshop CS (Adobe). Quantification ofT/B border localized B cells was done by counting cells that were within50 μm of the T/B border, as denoted by B220 counterstain, any cellslocalized in more central regions were considered follicular.

Electron Microscopy

Popliteal LNs were fixed in situ by footpad injection of 2% formaldehydeand 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4. TheLNs were excised and immersed in the same buffer overnight at 4° C.,washed in cacodylate buffer, and osmicated with 1% Osmium tetroxide/1.5%Potassium ferrocyanide (in water) for 1 hour at room temperature in thedark. After washing in water, samples were washed 3-4 times in 0.05 Mmaleate buffer pH 5.15. Samples were counterstained for 2 hours in 1%uranyl acetate in maleate buffer and washed three times in water.Samples were dehydrated by incubation for 15 minutes in dilutions ofethanol in water (70%-90%-100%), incubated in propylene oxide for 1hour, and transferred into Epon mixed 1:1 with propylene oxide RTovernight. Samples were moved to embedding mold filled with freshlymixed Epon, and heated for 24-48 hours at 60° C. for polymerization.Samples were analyzed on a Tecnai G2 Spirit BioTWIN electron microscopeat the Harvard Medical School EM facility.

Intravital Multiphoton Microscopy (MP-IVM) of the Popliteal LN

Naïve B cells were negatively selected by magnetic isolation using CD43beads (Miltenyi). VI10YEN B cells were labeled for 20 minutes at 37° C.with 10 μM 5-(and6-)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR;Invitrogen), C57BL/6 B cells were labeled for 25 minutes at 37° C. with10 μM 7-amino-4-chloromethylcoumarin (CMAC; Invitrogen). In someexperiments, labels were swapped between wildtype and VI10YEN B cells toexclude unspecific dye effects. 5-6×10⁶ B cells of each population weremixed and adoptively transferred by tail vein injection into C57BL/6recipient mice one day before analysis. In some experiments, recipientC57BL/6 mice had received an injection of 30 μl CLL into the hindfootpad 7-10 days before the experiment to eliminate SCS macrophages(Delemarre et al., 1990, J. Leukoc. Biol., 47:251; incorporated hereinby reference). Eighteen hours following adoptive B cell transfer,recipient mice were anaesthetized by intraperitoneal injection on ofketamine (50 mg/kg) and xylazine (10 mg/kg). The right popliteal LN wasprepared microsurgically for MP-IVM and positioned on a custom-builtmicroscope stage as described (Mempel et al., 2004, Nature, 427:154;incorporated herein by reference). Care was taken to spare blood vesselsand afferent lymph vessels. The exposed LN was submerged in normalsaline and covered with a glass coverslip. A thermocouple was placednext to the LN to monitor local temperature, which was maintained at36-38° C. MP-IVM was performed on a BioRad 2100 MP system at anexcitation wavelength of 800 nm, from a tunable MaiTai Ti:sapphire laser(Spectra-Physics). Fluorescently labeled VSV (20 ng in 20 n1) wasinjected through a 31 G needle into the right hind footpad of recipientmice concomitant to observation. For four-dimensional off-line analysisof cell migration, stacks of 11 optical x-y sections with 4 μm z spacingwere acquired every 15 seconds with electronic zooming to 1.8×-3×through a 20×/0.95 water immersion objective (Olympus). Emittedfluorescence and second harmonic signals were detected through 400/40nm, 450/80 nm, 525/50 nm, and 630/120 nm band-pass filters withnon-descanned detectors to generate three-color images. Sequences ofimage stacks were transformed into volume-rendered, four-dimensionaltime-lapse movies using Volocity software (Improvision). 3Dinstantaneous velocities were determined by semi-automated cell trackingwith Volocity and computational analysis by Matlab (Mathworks).Accumulation of cells at the SCS was determined by manual movie analysisperformed by blinded observers. Every 2 minutes, the VI10YEN B cells andpolyclonal B cells were counted at the SCS, in the superficial follicle(<50 μm distance from the SCS) and the deep follicle (>50 μm distancefrom the SCS), and ratios of VI10YEN/polyclonal B cells was expressedfor each compartment in the entire 30 minute movie.

Thoracic Duct Cannulation

For thoracic duct cannulation, mice received 200 μl olive oil p.o. 30minutes prior to cannulation to facilitate visualization of the lymphvessels. Animals were then anesthetized with xylazine (10 mg/kg) andketamine HCl (50 mg/kg). A polyethylene catheter (PE-10) was insertedinto the right jugular vein for continuous infusion (2 ml/hour) ofRinger's lactate (Abbott Laboratories, North Chicago, Ill.) containing 1U/ml heparin (American Pharmaceutical partners, Los Angeles, Calif.).Using a dissecting microscope, the TD was exposed through a leftsubcostal incision. Silastic® silicon tubing (0.012″ I.D., Dow Corning,Midland, USA) was flushed with heparinised (50 U/ml) phosphate-bufferedsaline (DPBS, Mediatech, Herndon, Va.), inserted into the cisterna chylithrough an approximately 0.3 mm incision and fixed with isobutylcyanoacrylate monomer (Nexaband®, Abbott Laboratories). The remainingpart of the tubing was exteriorized through the posterior abdominalwall. Subsequently, the abdominal incision was closed using a 6-0nonabsorbable running suture (Sofsilk, Tyco Healthcare Group, Norwalk,Colo.). Following a 30 minute equilibration of lymph flow, animals werefootpad injected with 108 pfu of VSV-IND and lymph samples werecollected on ice for 6 hours. Lymph and organs were taken after 6 hoursof thoracic duct lymph collection and plagued as described above. Lymphand organs were plagued as described above. In some experiments thedraining popliteal and paraaortic lymph nodes were surgically excisedand the surrounding lymph vessels cauterized to prevent lymph borneviral access to the blood.

Results and Discussion

Lymph nodes (LNs) prevent systemic dissemination of pathogens, such asviruses that enter the body's surfaces, from peripheral sites ofinfection. They are also the staging ground of adaptive immune responsesto pathogen-derived antigens (von Andrian and Mempel, 2003, Nat. Rev.Immunol., 3:867; and Karrer et al., 1997, J. Exp. Med., 185:2157; bothof which are incorporated herein by reference). It is unclear how virusparticles are cleared from afferent lymph and presented to cognate Bcells to induce antibody responses. Here, we identify a population ofCD11b⁺CD169⁺MHCII⁺ macrophages on the floor of the subcapsular sinus(SCS) and in the medulla of LNs that capture viral particles withinminutes after subcutaneous (s.c.) injection. SCS macrophagestranslocated surface-bound viral particles across the SCS floor andpresented them to migrating B cells in the underlying follicles.Selective depletion of these macrophages compromised local viralretention, exacerbated viremia of the host, and impaired local B cellactivation. These findings indicate that CD169⁺ macrophages have a dualphysiological function. They act as innate “flypaper” by preventing thesystemic spread of lymph-borne pathogens and as critical gatekeepers atthe lymph-tissue interface that facilitate B cell recognition ofparticulate antigens and initiate humoral immune responses.

We have investigated how virus particles that enter peripheral tissuesare handled within draining LNs. Hind footpads of mice were injectedwith fluorescently labeled UV-inactivated vesicular stomatitis virus(VSV), a cytopathic rhabdovirus that is transmittable by insect bites(Mead et al., 2000, Ann. N.Y. Acad. Sci., 916:437; incorporated hereinby reference) and elicits T-independent neutralizing B cell responses(Bachmann et al., 1995, Eur. J. Immunol., 25:3445; incorporated hereinby reference). Using multiphoton intravital microscopy (MP-IVM) inpopliteal LNs (Mempel et al., 2004, Nature, 427:154; incorporated hereinby reference) draining the injected footpad, we observed that VSVaccumulated in discrete patches on the SCS floor within minutes after scinjection, while the parenchyma and roof of the SCS remained free ofvirus (FIG. 11A). The viral deposits became progressively denser formingconspicuous irregular reticular patterns, which remained fixed in placefor hours.

To characterize the predilection sites for VSV binding in LNs, wereconstituted irradiated Act(EGFP) mice with wildtype bone marrow. Theresulting B6→Act(EGFP) chimeras expressed EGFP in non-hematopoieticcells, presumably lymphatic endothelial cells, on the SCS floor androof. Upon footpad injection of fluorescent VSV into C57BL/6→Act(EGFP)chimeras, viral particles flooded the SCS. Three hours later, unboundlumenal VSV had disappeared, but the SCS floor displayed prominentpatches of VSV that did not colocalize with EGFP⁺ cells, suggesting thatVSV was captured by hematopoietic cells (FIG. 11B). To characterize theputative VSV-capturing leukocytes, we performed electron microscopy onpopliteal LNs that were harvested 5 min after VSV injection (FIG. 11C).Bullet-shaped, electron-dense VSV particles were selectively bound todiscrete regions on the surface of scattered large cells that residedwithin the SCS or just below the SCS floor. VSV-binding cells that werelocated beneath the SCS floor were typically in contact with the lymphcompartment via protrusions that extended into the SCS lumen.

Ultrastructural studies of LNs have shown that the SCS contains manymacrophages (Clark, 1962, Am. J. Anat., 110:217; and Farr et al., 1980,Am. J. Anat., 157:265; both of which are incorporated herein byreference), so we hypothesized that the VSV-retaining cells belonged tothis population. Indeed, confocal microscopy of frozen LN sectionsobtained thirty minutes after footpad injection showed that VSVco-localized in the SCS with a macrophage marker, CD169/sialoadhesin(FIG. 11D). Using flow cytometry, we detected CD169 on approximately1%-2% of mononuclear cells (MNCs) in LNs, which uniformly co-expressedCD11b and MHC-II, indicating that the VSV-binding cells are indeedmacrophages (FIG. 12). Most CD169⁺ cells also expressed other macrophagemarkers, including CD68 and F4/80, while few expressed thegranulocyte/monocyte marker Gr-1. CD169⁺ cells also expressed CD11c, butat lower levels than CD11c^(high) conventional dendritic cells (DCs). Weconclude that intact virions enter the lymph within minutes aftertranscutaneous deposition and accumulate rapidly and selectively onmacrophages in the medulla and SCS of draining LNs.

To explore mechanisms for virus fixation, live VSV (20 μg containing2×10⁸ pfu) was injected into hind footpads and viral titers in drainingLNs were assessed 2 hours later. There was no defect in VSV retention indraining LNs of complement C3-deficient mice (FIG. 11E). DH-LMP2a mice,which lack secreted immunoglobulins, had reduced virus titers in spleen,but not in popliteal LNs (FIG. 11F). Therefore, VSV fixation in LNsoccurs via a mechanism distinct from that used by splenic marginal zonemacrophages, which require C3 and natural antibodies to captureblood-borne VSV (Ochsenbein et al., 1999, J. Exp. Med., 190:1165; andOchsenbein et al., 1999, Science, 286:2156; both of which areincorporated herein by reference). Conceivably, the VSV surfaceglycoprotein (VSV-G) may be recognized in LNs by macrophage-expressedcarbohydrate-binding scavenger receptors (Taylor et al., 2005, Ann. Rev.Immunol., 23:901; incorporated herein by reference), but the precisemechanism will require further investigation.

What are the consequences of viral capture by macrophages for virusdissemination and anti-viral immunity? To address this question, wedepleted LN-resident macrophages by footpad injection of clodronateliposomes (CLL; Delemarre et al., 1990, J. Leukoc. Biol., 47:251;incorporated herein by reference). At the dose used, sc injected CLLselectively eliminated macrophages in LNs draining the injection site,including the popliteal, inguinal and paraortic LNs (Delemarre et al.,1990, J. Leukoc. Biol., 47:251; incorporated herein by reference), whilemacrophages in distal LNs and spleen were spared (FIGS. 13 A, B). Amongthe different LN-resident CD11b⁺MHCII⁺ phagocytes, CLL preferentiallyremoved the CD169⁺ subset, whereas LYVE-1⁺ cells and conventional DCsremained unchanged. CLL-treated popliteal LNs had increased B cellnumbers and enlarged follicles 7 days after treatment, but othermorphological parameters, e.g. demarcation of the T/B border and SCSultrastructure remained unaltered (FIGS. 13 C-E).

Compared to untreated LNs, we recovered approximately 10-fold lowerviral titers from the draining LNs of CLL-treated mice (FIG. 11G),suggesting that macrophage depletion rendered lymph filtrationinefficient. Indeed, VSV titers were dramatically increased in blood,spleen, and non-draining LNs of CLL-treated mice. Viral disseminationfrom the injection site to the blood depended strictly on lymphdrainage, because circulating VSV was undetectable when virus wasinjected into footpads of mice that carried an occluding catheter in thethoracic duct (TD), even in CLL-treated mice. Viral titers were low, butdetectable in TD lymph fluid of untreated mice, but increasedsignificantly in CLL-treated animals (FIG. 11H). This indicates that theprincipal conduit for early viral dissemination from peripheral tissuesis the lymph, which is monitored by LN-resident, CLL-sensitivemacrophages that prevent the systemic spread of lymph-borne VSV.

This capture mechanism was not unique to VSV; CD169⁺SCS macrophages alsoretained adenovirus (AdV; FIGS. 14 A-C) and vaccinia virus (VV, FIG.14D), indicating that macrophages act as guardians against manystructurally distinct pathogens. In contrast, virus-sized latex beads(200 nm) were poorly retained in the SCS after footpad injection (FIG.14E). Thus, SCS macrophages discriminate between lymph-borne viruses andother particles of similar size. Fluorescent VSV, AdV and VV alsoaccumulated in the medulla of draining LNs, where they were not onlybound by CD169^(low) cells (FIG. 11D) but also by CD169⁻LYVE-1⁺lymphatic endothelial cells (FIGS. 14 C, D). This was corroborated inCLL-treated LNs, where VSV accumulated exclusively on medullary LYVE-1⁺cells (FIG. 15).

Next, we examined how captured VSV is recognized by B cells. PoplitealLNs contain rare B cells in the SCS lumen (FIG. 16A), but we found noevidence for virus-binding lymphocytes within the SCS on electronmicrographs. Instead, viral particles were presented to B cells withinsuperficial follicles by macrophages that extended across the SCS floor.Following injection of either VSV (FIG. 17A) or AdV (FIGS. 16 B-E),virions were readily detectable at B cell-macrophage interfaces for atleast 4 hours. This suggested that SCS macrophages shuttle viralparticles across the SCS floor for presentation to B cells. Transcytosisseemed unlikely, because the few vesicles containing VSV in SCSmacrophages showed evidence of viral degradation. In addition, we didnot detect substantial motility of virus-binding macrophages by MP-IVM,at least during the first 6 hours after challenge. Therefore, viralparticles most likely reached the LN parenchyma by moving along themacrophage surface. Of note, VSV and other antigens are also presentedto B cells by DCs immigrating from peripheral locations (Ludewig et al.,2000, Eur. J. Immunol., 30:185; and Qi et al., 2006, Science, 312:1672;both of which are incorporated herein by reference), but footpad-derivedDCs are not likely to play a role during these very early events,because their migration into popliteal LNs takes much longer. Weconclude that the SCS floor is not insurmountable for lymph-borneviruses; CD169⁺ macrophages appear to act as gatekeepers andfacilitators of viral translocation and presentation to B cells.

Next, we explored how naive B cells respond to viral encounter using twoVSV serotypes, Indiana (VSV-IND) and New Jersey (VSV-NJ) (FIG. 18; Roostet al., 1996, J. Immunol. Methods, 189:233; incorporated herein byreference). We compared wildtype B cells to B cells from VI10YEN mice,which express a VSV-IND-specific B cell receptor that does not bindVSV-NJ (Hangartner et al., 2003, Proc. Natl. Acad. Sci., USA, 100:12883;incorporated herein by reference). By contrast, a small fraction (2%-5%)of wildtype B cells bound both serotypes without being activated. Thismight reflect low-affinity reactivity with VSV-G or indirectinteractions, e.g. via complement (Rossbacher and Shlomchik, 2003, J.Exp. Med., 198:591; incorporated herein by reference). To assess in vivoresponses, differentially labeled wildtype and VI10YEN B cells wereadoptively transferred and allowed to home to LN follicles. FluorescentUV-inactivated virus was then injected into footpads and popliteal LNswere recorded by MP-IVM about 5-35 minutes later. In virus-free LNs orafter injection of VSV-NJ, VI10YEN and control B cells displayed thesame distribution (FIGS. 17 B-C). In contrast, upon VSV-IND injectionVI10YEN cells rapidly accumulated below and at the SCS floor. There wasno difference in baseline B cell motility and distribution betweenCLL-treated and untreated LNs, suggesting that VSV-specific B cells areequally likely to probe the SCS in both conditions. However, inCLL-treated LNs, fluorescent virus was not retained in the SCS andVI10YEN B cells failed to congregate in that region, indicating that SCSmacrophages are essential for both events (FIG. 17B).

To rigorously quantify VI10YEN B cell distribution, LNs were harvested30 minutes after VSV challenge and analyzed by confocal microscopy.While the entire follicular VI10YEN population retained its overalldistribution (FIG. 17D), the subset of cells residing ≦50 μm below theSCS shifted toward the SCS in VSV-IND, but not VSV-NJ containing LNs(FIG. 17E). It seems unlikely that VI10YEN B cells redistributed to theSCS because of chemoattractant signals, since unresponsive polyclonal Bcells express the same chemoattractant receptors. More likely, therandom contacts of motile VI10YEN cells with macrophage-bound VSV-INDtriggered a BCR-dependent “stop signal” (Okada et al., 2005, PLoS Biol.,3:e150; incorporated herein by reference): Short-term exposure toVSV-IND activates LFA-1 and/or α4 integrins (Dang and Rock, 1991, J.Immunol., 146:3273; incorporated herein by reference) on VI10YEN Bcells, resulting in adhesion to the respective ligands, ICAM-1 andVCAM-1, which are both expressed in the SCS (FIG. 19). Additionally,VSV-IND bound to SCS macrophages may provide a substrate for VI10YEN Bcell adhesion directly via the BCR.

To investigate how captured virions are processed upon detection by Bcells, we tested B cells from VI10YENxMHCII-EGFP mice, which allowed usto visualize endocytosed VSV co-localizing with endosomal MHC-II as anindicator of B cell priming (Vascotto et al., 2007, Curr., Opin.,Immunol., 19:93; incorporated herein by reference). Within 30 minutesafter injection, VI10YENxMHCII-EGFP B cells in the superficial folliclehad extensively internalized VSV-IND, but not VSV-NJ particles (FIGS. 20A, B). Virus-carrying VSV-specific B cells were infrequent, butdetectable in deep follicles. These cells may have acquired virions fromrare polyclonal B cells that carried VSV on their surface, or maycorrespond to VI10YEN cells which failed to arrest at the SCS afteracquiring VSV-IND.

While our histological findings demonstrate that intact virions arepreferentially detected and acquired by B cells in the SCS andsuperficial follicle, MP-IVM measurements of B cell motility revealedbroader antigen dissemination. After VSV-IND injection, VI10YEN cellsexhibited a rapid drop in velocity throughout the entire B follicle,(FIG. 21). This was equally observed in CLL-treated and control LNs,indicating that viral antigen reached B cells independent ofmacrophages. This antigenic material was most likely composed of freeviral protein, an inevitable by-product of natural infections. Indeed,purified supernatant of our VSV stocks induced a potent calcium flux inVI10YEN B cells (FIG. 18E). Small lymph-borne proteins are known todiffuse rapidly into follicles and activate cognate B cells (Pape etal., 2007, Immunity, 26:491; incorporated herein by reference).Accordingly, injection of viral supernatant suppressed the motility offollicular VI10YEN B cells without inducing their accumulation at theSCS, indicating that free VSV-G was contained and active within theviral inoculum. This can explain the macrophage-independentpan-follicular effect of VSV-IND injection.

To determine the kinetics of VI10YEN B cell activation upon viralencounter, we measured common activation markers (FIG. 22). Thecostimulatory molecule CD86 was first up-regulated 6 hours after VSV-INDchallenge. CD69 was induced more rapidly, but also on polyclonal Bcells, presumably by pleiotropic IFN-α signaling (Barchet et al., 2002,J. Exp. Med., 195:507; and Shiow et al., 2006, Nature, 440:540; both ofwhich are incorporated herein by reference). Surface IgM (FIGS. 20 C, D)was down-regulated as early as 30 minutes after challenge reaching amaximum within 2 h when >70% of VI10YEN cells were BCR^(low/neg).Therefore, BCR internalization provided the earliest specific readoutfor virus-specific B cell activation. Remarkably, VI10YEN B cells inCLL-treated LNs failed to downregulate their BCR during the first 2hours after subcutaneous injection of 20 mg VSV-IND (FIG. 20E),indicating that SCS macrophages are necessary for efficient earlypresentation of captured virions to B cells.

Primed B cells eventually solicit help from CD4⁺ T cells (Vascotto etal., 2007, Curr., Opin., Immunol., 19:93; incorporated herein byreference) for class switch recombination and germinal center formation.To contact T cells, newly activated B cells migrate toward the T/Bborder (Okada et al., 2005, PLoS Biol., 3:e150; and Reif et al., Nature,416:94; both of which are incorporated herein by reference). Thismechanism operated efficiently in macrophage-sufficient mice; mostVI10YEN B cells redistributed to the T/B border within 6 h after footpadinjection of as little as 40 ng VSV-IND (FIGS. 20 F, H and 23). Bycontrast, a 100-fold higher viral dose was needed to elicit fullredistribution of VI10YEN B cells in CLL-treated mice (FIGS. 20 G, H).By 12 hours after injection, most VSV-specific cells reached the T-Bborder, irrespective of the injected dose. Thus, even without SCSmacrophages follicular B cells are eventually activated by VSV-derivedantigen, albeit less efficiently.

In conclusion, we demonstrate a dual role for CD169⁺ macrophages in LNs:they capture lymph-borne viruses preventing their systemic disseminationand they guide captured virions across the SCS floor for efficientpresentation and activation of follicular B cells.

Example 2 Exemplary Lipid-Based Vaccine Nanotechnology Architectures

Liposome Nanocarriers

In some embodiments, small liposomes (10 nm-1000 nm) are manufacturedand employed to deliver, in some embodiments, one or multipleimmunomodulatory agents to cells of the immune system (FIG. 3). Ingeneral, liposomes are artificially-constructed spherical lipidvesicles, whose controllable diameter from tens to thousands of nmsignifies that individual liposomes comprise biocompatible compartmentswith volume from zeptoliters (10⁻²¹ L) to femtoliters (10⁻¹⁵ L) that canbe used to encapsulate and store various cargoes such as proteins,enzymes, DNA and drug molecules. Liposomes may comprise a lipid bilayerwhich has an amphiphilic property: both interior and exterior surfacesof the bilayer are hydrophilic, and the bilayer lumen is hydrophobic.Lipophilic molecules can spontaneously embed themselves into liposomemembrane and retain their hydrophilic domains outside, and hydrophilicmolecules can be chemically conjugated to the outer surface of liposometaking advantage of membrane biofunctionality.

In certain embodiments, lipids are mixed with a lipophilicimmunomodulatory agent, and then formed into thin films on a solidsurface. A hydrophilic immunomodulatory agent is dissolved in an aqueoussolution, which is added to the lipid films to hydrolyze lipids undervortex. Liposomes with lipophilic immunomodulatory agents incorporatedinto the bilayer wall and hydrophilic immunomodulatory agents inside theliposome lumen are spontaneously assembled.

Nanoparticle-Stabilized Liposome Nanocarriers

In some embodiments, nanoparticle-stabilized liposomes are used todeliver one or a plurality of immunomodulatory agents to cells of theimmune system (FIG. 4). When small charged nanoparticles approach thesurface of liposomes carrying either opposite charge or no net charge,electrostatic or charge-dipole interaction between nanoparticles andmembrane attracts the nanoparticles to stay on the membrane surface,being partially wrapped by lipid membrane. This induces local membranebending and globule surface tension of liposomes, both of which enabletuning of membrane rigidity. This aspect is significant for vaccinedelivery using liposomes to mimic viruses whose stiffness depends on thecomposition of other biological components within virus membrane.Moreover, adsorbed nanoparticles form a charged shell which protectsliposomes against fusion, thereby enhancing liposome stability. Incertain embodiments, small nanoparticles are mixed with liposomes undergentle vortex, and the nanoparticles stick to liposome surfacespontaneously.

Liposome-Polymer Nanocarrier

In some embodiments, liposome-polymer nanocarriers are used to deliverone or a plurality of immunomodulatory agents to cells of the immunesystem (FIG. 5). Instead of keeping the liposome interior hollow,hydrophilic immunomodulatory agents may be encapsulated. FIG. 3 showsliposomes that are loaded with di-block copolymer nanoparticles to formliposome-coated polymeric nanocarriers, which have the merits of bothliposomes and polymeric nanoparticles, while excluding some of theirlimitations. In some embodiments, the liposome shell can be used tocarry lipophilic or conjugate hydrophilic immunomodulatory agents, andthe polymeric core can be used to deliver hydrophobic immunomodulatoryagents. In certain embodiments, pre-formulated polymeric nanoparticles(40 nm-1000 nm) are mixed with small liposomes (20 nm-100 nm) undergentle vortex to induce liposome fusion onto polymeric nanoparticlesurface.

Nanoparticle-Stabilized Liposome-Polymer Nanocarriers

In some embodiments, nanoparticle-stabilized liposome-polymernanocarriers are used to deliver one or a plurality of immunomodulatoryagents (FIG. 6). By adsorbing small nanoparticles (1 nm-30 nm) to theliposome-polymer nanocarrier surface, the nanocarrier has not only themerit of both aforementioned nanoparticle-stabilized liposomes (FIG. 4)and aforementioned liposome-polymer nanoparticles (FIG. 5), but alsotunable membrane rigidity and controllable liposome stability.

Liposome-Polymer Nanocarriers Comprising Reverse Micelles

In some embodiments, liposome-polymer nanocarriers containing reversemicelles are used to deliver one or a plurality of immunomodulatoryagents (FIG. 7). Since the aforementioned liposome-polymer nanocarriers(FIGS. 5 and 6) are limited to carry hydrophobic immunomodulatory agentswithin polymeric nanoparticles, here small reverse micelles (1 nm-20 nm)are formulated to encapsulate hydrophilic immunomodulatory agents andthen mixed with the di-block copolymers to formulate polymeric core ofliposomes.

In certain embodiments, a hydrophilic immunomodulatory agent to beencapsulated is first incorporated into reverse micelles by mixing withnaturally derived and non-toxic amphiphilic entities in a volatile,water-miscible organic solvent. The resulting biodegradablepolymer-reverse micelle mixture is combined with a polymer-insolublehydrophilic non-solvent to form nanoparticles by the rapid diffusion ofthe solvent into the non-solvent and evaporation of the organic solvent.Reverse micelle contained polymeric nanoparticles are mixed with lipidmolecules to form the aforementioned liposome-polymer complex structure(FIG. 5).

Nanoparticle-Stabilized Liposome-Polymer Nanocarriers Comprising ReverseMicelles

In some embodiments, nanoparticle-stabilized liposome-polymernanocarriers containing reverse micelles are used to deliver one or aplurality of immunomodulatory agents (FIG. 8). By adsorbing smallnanoparticles (1 nm-30 nm) to a liposome-polymer nanocarrier surface,the nanocarrier has not only the merit of both aforementionednanoparticle-stabilized liposomes (FIG. 4) and aforementioned reversemicelle contained liposome-polymer nanoparticles (FIG. 7), but alsotunable membrane rigidity and controllable liposome stability.

Lipid Monolayer-Stabilized Polymeric Nanocarrier

In some embodiments, lipid monolayer stabilized polymeric nanocarriersare used to deliver one or a plurality of immunomodulatory agents (FIG.9). As compared to aforementioned liposome-polymer nanocarrier (FIGS.5-8), this system has the merit of simplicity in terms to both agentsand manufacturing. In some embodiments, a hydrophobic homopolymer canform the polymeric core in contrast to the di-block copolymer used inFIGS. 5-8, which has both hydrophobic and hydrophilic segments.Lipid-stabilized polymeric nanocarriers can be formed within one singlestep instead of formulating polymeric nanoparticle and liposomeseparately followed by fusing them together.

In certain embodiments, a hydrophilic immunomodulatory molecule is firstchemically conjugated to a lipid headgroup. The conjugate is mixed witha certain ratio of unconjugated lipid molecules in an aqueous solutioncontaining one or more water-miscible solvents. A biodegradablepolymeric material is mixed with the hydrophobic immunomodulatory agentsto be encapsulated in a water miscible or partially water miscibleorganic solvent. The resulting polymer solution is added to the aqueoussolution of conjugated and unconjugated lipid to yield nanoparticles bythe rapid diffusion of the organic solvent into the water andevaporation of the organic solvent.

Lipid Monolayer-Stabilized Polymeric Nanocarrier Comprising ReverseMicelles

In some embodiments, lipid monolayer stabilized polymeric nanoparticlescomprising reverse micelles are used to deliver one or a plurality ofimmunomodulatory agents (FIG. 10). Since the aforementionedlipid-stabilized polymeric nanocarriers (FIG. 9) are limited to carryhydrophobic immunomodulatory agents, here, small reverse micelles (1nm-20 nm) are formulated to encapsulate hydrophilic immunomodulatoryagents and mixed with biodegradable polymers to form polymericnanocarrier core.

Example 3 In Vivo Targeting of SCS-Mph Using Fc Fragments from Human IgG

Fluorescent unmodified control nanoparticles (top panel, FIG. 24A) or Fcsurface-conjugated targeted nanoparticles (middle and lower panel, FIG.24A) were injected into footpads of anesthetized mice, and the drainingpopliteal lymph node was excised 1 hour later and single-cellsuspensions were prepared for flow cytometry. Targeted nanoparticleswere also injected into mice one week after lymph node macrophages hadbeen depleted by injection of clodronate-laden liposomes (lower panel,FIG. 24A). The cell populations in gates were identified asnanoparticle-associated macrophages based on high expression of CD11b.These results indicate that (i) nanoparticle binding depends on thepresence of clodronate-sensitive macrophages and (ii) targetednanoparticles are bound to twice as many macrophages as controlnanoparticles.

The Panels on the right of FIG. 24 show fluorescent micrographs offrozen lymph node sections after injection of blue fluorescent control(top panel, FIG. 24A) or targeted (middle and lower panels, FIG. 24A)nanoparticles. Sections were counter-stained with anti-CD169 and amarker that identifies either the medulla (in top and bottom panel, FIG.24A) or B cells (in middle panel, FIG. 24A). At one hour afternanoparticle injection most control particles are found in the medulla(top, FIG. 24A), while targeted nanoparticles colocalise withCD169+SCS-Mph adjacent to B cell follicles (middle, FIG. 24A). At 24hours after injection, discrete cell-sized accumulations of targetednanoparticles are seen in the cortical region between the SCS and themedulla, suggesting uptake and transport by migratory dendritic cells.

Mice were injected i.v. with red fluorescent B cells and in a footpadwith a 1:1 mixture of control and Fc targeted nanoparticles. 24 hourslater, when some of the transferred B cells had migrated into B cellfollicles, the draining popliteal lymph node was excised and sectionedfor confocal microscopy and quantitative image analysis of green:bluefluorescent ratios. The subcapsular sinus (SCS) region contained similarlevels of blue and green nanoparticles (cells encircled on the right,FIG. 24B), while green fluorescence associated with Fc-targetednanoparticles was about twice higher in the SCS. There were alsoprominent accumulations of green nanoparticles within B folliclesdelineated by scattered red B cells. These regions have thecharacteristic size, shape, and distribution of follicular dendriticcells (FDC), which like macrophages and dendritic cells are known toexpress abundant Fc receptors.

Example 4 Antigen-Bearing Targeted Nanoparticles are Highly Immunogenicand Induce High Antibody Titers

Groups of mice (5/group) were immunized with: UV-inactivated vesicularstomatits virus (VSV, serotype Indiana) or with the purified immunogenicenvelope glycoprotein (VSV-G) of VSV. VSV-G was either given in solubleform mixed with alum or conjugated to non-targeted or targeted (withsurface immobilized human Fc) PLGA nanoparticles with or without alum asan adjuvant. The dose of free VSV-G was estimated to be ˜10-fold higherthan the dose of VSV-G delivered with nanoparticles. Mice received abooster injection at day 55 after the primary immunization, and serumwas obtained after 10 weeks and tested for neutralization ofVSV-mediated plaque formation on Vero cells. Results show titers as thehighest serum dilution that blocked plaque formation by at least 50%.Each symbol reflects the neutralizing anti-VSV titer in one mouse. Thegroup of mice immunized with VSV-G presented on Fc-targetednanoparticles generated significantly higher neutralizing anti-VSVtiters than any other group (the two animals with the highest titers inthat group completely neutralized plaque formation at the highestdilution tested, so actual titers may have been even higher).

The induced immune response elicited by nanoparticle (NP) vaccinesconfers potent protection from a lethal dose of VSV. While allvaccinated groups showed some protection, only the group that receivedVSV-G conjugates to Fc-targeted NPs plus alum showed 100% protectionfrom lethal infection. Recipients of free VSV-G (VSV-G+alum) received˜10-fold more antigen than animals that were given VSV-G conjugated tonanoparticles. As a negative control, one group of mice receivedFc-targeted nanoparticles (NP-Fc) without VSV-G, which did not conferprotection.

Example 5 In Vivo T Cell Activation by Immunomodulatory Nanoparticles

C57BL6J mice were injected i.v. with CF SE-labeled CD4 T cells fromOT-II donor mice, which express a transgenic TCR specific for chickenovalbumin (OVA) presented in MHC class II. Subsequently, immunizationexperiments were performed by injecting one footpad with free OVA orwith nanoparticles composed of either PLA or PLGA that encapsulated anequivalent amount of OVA as a model antigen. All antigenic mixtures alsocontained CpG (a TLR9 agonist) as an adjuvant. The animals wereinjected, sacrificed three days after immunization, and OT-II T cellactivation was assessed by flow cytometry in single-cell suspensionsfrom different tissues.

Unstimulated 5,6-carboxy-succinimidyl-fluorescein-ester (CFSE)-labeled Tcells do not divide and, therefore, carry an uniformly highconcentration of CFSE resulting in a single narrow peak of brightlyfluorescent cells. By contrast, activated T cells divide and in theprocess split the fluorescent dye evenly between the two daughter cellsresulting in an incremental decrease in fluorescence intensity upon eachsuccessive division. Thus, the greater the left shift in CFSE,fluorescence the stronger T cells were activated. The results indicatethat: (i) nanoparticle-encapsulated antigen generated a more potent CD4T cell response than free antigen in the draining popliteal lymph node(popLN, top row); (ii) only nanoparticles, but not free OVA inducedlocal T cell proliferation in distal lymphoid tissues, including thebrachial lymph node (middle row) and the spleen (bottom row). Inrecipients of free OVA, the brachial LN and spleen contained onlyundivided cells or cells with very low CFSE content. The latterpopulation does not indicate local T cell activation but migration of Tcells that were activated elsewhere.

C57BL6J mice were injected i.v. with CFSE-labeled CD8 T cells from OT-Idonor mice, which express a transgenic T cell receptor (TCR) specificfor chicken ovalbumin (OVA) presented in MHC class I. The experimentalprotocol was otherwise identical as immediately described above.

C57BL6J mice were injected i.v. with CFSE-labeled CD8 T cells from OT-Idonor mice as above. However, in this experiment CL097, animidazoquinoline compound that activates TLR-7 and TLR-8, was used asadjuvant and different methods of adjuvant delivery were tested. T cellactivation in this case was assessed by counting the total number ofOT-I T cells in the draining popliteal lymph node three days afterfootpad injection of either free OVA (1 μg or 100 ng) mixed with freeadjuvant (160 ng). All animals that received nanoparticles were given100 ng OVA with or without 160 ng CL097. Material that was encapsulatedwithin nanoparticles, but not covalently attached to the PLA polymer isshown in [ ]. Covalent linkage of CL097 to PLA is identified byhyphenation. Materials that were mixed in free form within the samecompartment are separated by “+”. These results revealed a markedincrease in CD8 T cell proliferation in animals that receivedencapsulated OVA in nanoparticles in which the adjuvant was covalentlylinked to the excipient.

Example 6 Formation of Nanoparticles

PLA-PEG-COOH (3 mg/ml in DCM) was mixed with PLA-R848 (3 mg/ml in DCM)or R848 (15.7 mg/ml in DCM/DMSO (5%)). Added 500 μl of PLA-PEG-COOHsolution to 500 μl PLA-R848, vortex, added 500 μl of OVA (5 mg/ml inwater) or SIINFEKL peptide (2 mg/ml), sonicated (15 sec, 50 Amplitude)using SINOMIX (sonicator S-4000), added 2 ml of PVA solution (1% w/v inwater), sonicated (15 sec, 50 Amplitude) and dropped the solution into40 ml of water. Let stir for 2 hours, washed 3 times with water and 1time with PBS. For encapsulated R848 formulation, added 500 μl ofPLA-PEG-COOH solution to 400 μl free PLA (3.75 mg/ml) and 100 μl ofR848, vortexed, added 500 μl of OVA (5 mg/ml in water) or SIINFEKLpeptide (2 mg/ml), sonicated (15 sec, 50 Amplitude) added 2 ml of PVAsolution (1% w/v in water), sonicated (15 sec, 50 W) and dropped thesolution into 40 ml of water. Let stir for 2 hours, washed 3 times withwater and 1 time with PBS.

Example 7 Nicotine Nanoparticles

Nicotine-nanoparticle (nicotine-NP) formation. FIGS. 32 a and 32 b showthe a depiction of the process of forming nicotine-NP.Nicotine-nanoparticles were formed by combining varying ratios of thefollowing components: PLA-PEG amphiphilic block copolymer (3.5 kDA, endfunctionalized with —COOH); PLA-PEG amphiphilic block copolymerend-functionalized (i.e., conjugated) with nicotine; and PLA-adjuvantconjugate. In FIG. 32 a, phase A (water) and phase B (solvent) arecombined along with the components that form the nanoparticles. Themixture is sonicated or homogenized (step 10), forming primary (w/o)emulsion 20. A further aqueous phase (PVA) is added and the mixture issonicated or homogenized (step 30), forming secondary (w/o/w) emulsion40. Secondary emulsion 40 is allowed to incubate to allow solventevaporation (step 50). Nanoparticles 60 are formed in this manner. Thenanoparticles formed with an average size of about 250 nm and an a molaramount of nicotine (per particle) within the range of 1-100,000. Examplemixing ratios are shown in FIG. 32 b. Additional mixing rations (e.g.,50% nicotine-copolymer with 10%, 20%, 30%, or 40% PLA-adjuvant, thebalance being copolymer, or 40% nicotine-copolymer with similar ratio ofPLA-adjuvant, or 30% nicotine-copolymer with similar ratios ofPLA-adjuvand and copolymer, etc.) are

Example 8 Conjugate Formation

Cotinine-PEG-cotinine conjugates were formed according to the followingreaction equation, using doubly end-functionalized PEG:

Reduction of cotinine-PEG-cotinine with lithium aluminum hydride allowedformation of HO-PEG-Nicotine:

Similarly, copolymers of PLA and PEG, conjugated to nicotine, wereprepared using HO-PEG-nicotine as an initiator in a ring-openingpolymerization according to the following reaction:

Nicotine-NP were prepared using 50% PLA-PEG-nicotine and 50% PLA andadministered to mice via subcutaneous (sc) injection. The draining LNswere harvested 1 hour after sc injection and the sections were stainedwith APC-B220 to label B cells (in gray) and anti-nicotine Ab (cloneHB-9123) followed by Alexa568-anti-mouse IgG (in red). Fluorescence datashows that nicotine-NP accumulate in the SCS of the draining LN 1 hourafter sc injection.

Example 9 Anti-Nicotine IgG in Immunized Mice

In a first series of experiments, groups of C57BL6 mice (4-5) wereimmunized with nicotine immunonanotherapeutic compositions on day 0 andboosted after 2, 4 and 8 weeks. Ab titers were measured by ELISA andcompared to a standard curve from an anti-nicotine MAb to calculateconcentrations. One group of mice received OVA-specific OT-II T cells(5×10⁵ IV) prior to vaccination to boost T cell help. Data are shown inFIG. 33. Curves are identified by the immunization formulation. Forexample, the data labeled “nicotine+R848+OVA” indicates mice that wereimmunized with a formulation containing free nicotine, free R848, andfree OVA. The data labeled “PLA-nicotine[OVA]” indicates mice immunizedwith a formulation containing PLA-nicotine nanoparticles withencapsulated OVA.

In a second series of experiments, additional nicotine titers weredetermined and are shown in FIG. 34. The additional data show initial (3week) titers. The protocol in terms of dose and timing of injections wasthe same as for the first series. However, several new particleformulations were tested to assess: a) whether previously frozennanoparticles (containing 50% nicotine-PEG-PLA) retain theirimmunogenicity; b) how the content of nicotine-PEG-PLA affects antibodytiters; and c) whether the nanoparticles (50% nicotine-PEG-PLA) work inMHC class II deficient mice. The formulations are as follows: (1) NPhaving 50% nicotine-PEG-PLA and 50% PLA-R848, with encapsulated OVA,used as prepared (i.e., without freezing); (2) NP having 50%nicotine-PEG-PLA and 50% PLA-R848, with encapsulated OVA, used afterovernight freezing and subsequent thawing; (3) NP having 5%nicotine-PEG-PLA, 45% PLA-PEG, and 50% PLA-R848; (4) NP having 25%nicotine-PEG-PLA, 25% PLA-PEG, and 50% PLA-R848; (5) NP having 75%nicotine-PEG-PLA and 25% PLA-R848, wherein the formulation was given attwice the regular dose to keep the R848 dose constant; (6) NP having 50%nicotine-PEG-PLA and 50% PLA, with encapsulated OVA, wherein free R848was mixed into the formulation immediately prior to injection; (7) NPhaving 50% nicotine-PEG-PLA nanoparticles and 50% PLA-R848 withencapsulated OVA, wherein the mice received OVA-specific OT-II T cellsprior to vaccination; (8) NP having 50% nicotine-PEG-PLA and 50%PLA-R848 with encapsulated OVA, MHC class II deficient mice; and (9) NPhaving 50% nicotine-PEG-PLA and 50% PLA with encapsulated OVA, MHC classII deficient mice. Due to differences in ELISA sensitivity, the absolutemagnitude of titers in this series can't be compared directly to thefirst series of data. The following conclusions were reached: a)previously frozen and thawed NPs were still immunogenic; b) earlyantibody titers were similar in mice receiving NPs with nicotine-PEG-PLAcontent ranging from 5 to 50% (with always the same 50% amount ofR848-PLA), but lower when NPs with 75% nicotine-PEG-PLA was used; c) ahumoral response to nicotine-NPs was induced in MHC-II deficient mice,indicating the ability to induce a T-independent IgG response.

The data in FIGS. 33 and 34 show that free nicotine (i.e., notconjugated), even when administered along with PLA-R848, R848, and/orOVA, does not elicit the production of nicotine antibodies. However,nicotine nanoparticles do elicit such production. Even in the absence ofT cell help, the nicotine nanoparticles described herein cause asubstantial antibody production.

Example 10 Nanoparticle Targeting

I. Subcapsular Sinus Macrophage (SCS-Mph) Targeting by LiposomeContaining Ligands

Liposome preparation and characterization. Small unilamellar liposomeswere prepared by vesicle extrusion method. Lipids dissolved inchloroform were dried under a stream of nitrogen followed by 3 hoursvacuum desiccation. Next, the lipids were rehydrated in PBS buffer.After five freeze-thaw cycles, the liposomes were extruded twenty timesthrough a polycarbonate filter (Whatman) containing 100-nm pores.Liposomes prepared by this method were characterized by dynamic lightscattering with a Particle Size Analyzer (Brookhaven Instruments).

Liposomes tested for SCS-Mph targeting. (1) Several samples of liposomeswere prepared containing1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),monosialoganglioside GM3, and N-4-nitrobenzo-2-oxa-1,3-diazolephosphatidylethanolamine (NBD-PE). The density of GM3 across samplesvaried from ˜10 to ˜30 mol %. The lipid concentration is around 1 mg/mLand the average liposome size is about 110 nm. The targeting data showedthat higher GM3 density could result in better targeting. Specifically,the data showed accumulation of the liposomes on SCS Mphs in thedraining LN one hour after sc injection. (2) Liposomes containing onlyPOPC and NBD-PE were also prepared, as a control. No targeting wasobserved under the conditions tested. The lipid concentration andliposome size are similar to (1).

II. SCS-Mph Targeting by Polymer Nanoparticle Containing Ligands

1. Nanoparticle (NP) preparation and characterization. NPs were preparedby nano-precipitation method. 100 uL polymer (PLA-PEG-COOH 5 mg/mL inacetonitrile) was added dropwise into 500 uL DI water. The solution wasstirred at 400 rpm for 2 hours and washed twice with DI water and onetime with PBS by using Amicon tubes (100 KDa). The final concentrationof NP solution was about 1 mg/mL. The average particle size (˜74 nm) wasdetermined by dynamic light scattering with a Particle Size Analyzer(Brookhaven Instruments).

2. Ligand conjugation. Ligands including lysozyme, protein G,anti-CD169, and sialyllactose, were conjugated to the NP surface throughEDC/NHS reaction. After the polymer nanoprecipitation, the NP solutionwas washed twice with DI water. The surface carboxylic acid groups werethen activated by incubation with EDC/NHS (15 mg/mL) solution for 1hour. Excess EDC and NHS were washed away with DI water and then PBSbuffer. The NP solution was then mixed with ligand solution (the molarratio of polymer vs. ligand is 1:1 for proteins and 1:2 forsialyllactose) for 2 hours, and washed with PBS buffer for three times.For PLA-PEG-OCH3 targeting experiment, the NP was prepared with the sameprocedure as described in (1). For NP imaging, 10% PLA-Alexa Fluor 647was blended with PLA-PEG polymer in the nanoprecipitation experiment.

Conclusion: The confocal fluorescence images showed NP—OCH3 andNP-antiCD169 could bind to SCS-Mph, and a little accumulation ofNP-sialyllactose in the SCS. No binding of NP-lysozyme and NP-protein Gto SCS-Mph was observed under the particular set of experimentalconditions.

III. SCS-Mph Targeting by Polystyrene Beads Containing Ligands

1. Polystyrene beads and ligand conjugation. The NeutrAvidin labeledbeads (FluoSpheres, 0.01 g/mL) were purchased from Invitrogen. Theaverage size is around 200 nm. Ligands including biotinylated anti-CD169and biotinylated oligomer G (12 mer) were linked to NeutrAvidin labeledbeads by incubation for 2 hours.

2. Results. Both anti-CD169 and oligomer G conjugated beads accumulatedon SCS-Mphs.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention, described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the appended claims.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the appended claims.

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Thus, for example, reference to “a nanoparticle” includes aplurality of such nanoparticle, and reference to “the cell” includesreference to one or more cells known to those skilled in the art, and soforth. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process. Furthermore, it is to be understood that theinvention encompasses all variations, combinations, and permutations inwhich one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim. For example, any claim that is dependent on another claim can bemodified to include one or more limitations found in any other claimthat is dependent on the same base claim. Furthermore, where the claimsrecite a composition, it is to be understood that methods of using thecomposition for any of the purposes disclosed herein are included, andmethods of making the composition according to any of the methods ofmaking disclosed herein or other methods known in the art are included,unless otherwise indicated or unless it would be evident to one ofordinary skill in the art that a contradiction or inconsistency wouldarise.

Where elements are presented as lists, e.g., in Markush group format, itis to be understood that each subgroup of the elements is alsodisclosed, and any element(s) can be removed from the group. It shouldit be understood that, in general, where the invention, or aspects ofthe invention, is/are referred to as comprising particular elements,features, etc., certain embodiments of the invention or aspects of theinvention consist, or consist essentially of, such elements, features,etc. For purposes of simplicity those embodiments have not beenspecifically set forth in haec verba herein. It is noted that the term“comprising” is intended to be open and permits the inclusion ofadditional elements or steps.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., anyimmunomodulatory agent, any targeting moiety, any immunostimulatoryagent, any antigen presenting cell, any vaccine nanocarrierarchitecture, any microorganism, any method of administration, anyprophylactic and/or therapeutic application, etc.) can be excluded fromany one or more claims, for any reason, whether or not related to theexistence of prior art.

The publications discussed above and throughout the text are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior disclosure.

1. A composition comprising: (1) synthetic nanocarriers having at leastone surface, wherein a first surface of the synthetic nanocarrierscomprises an immunofeature surface comprising a plurality of nicotinemoieties; and (2) a pharmaceutically acceptable excipient.
 2. Thecomposition of claim 1, wherein the composition further comprises animmunostimulatory agent, wherein the immunostimulatory agent: (i) isassociated with the immunofeature surface: (ii) is associated with asecond surface of the nanocarrier; or (iii) is encapsulated within thenanocarrier.
 3. The composition of claim 1, wherein the syntheticnanocarriers are capable of activating CD4+ T cells, NKT cells, or bothwhen administered to a human.
 4. The composition of claim 1, wherein thesynthetic nanocarriers are capable of stimulating production ofanti-nicotine IgG antibodies when administered to a human.
 5. Thecomposition of claim 1, wherein the synthetic nanocarriers are capableof eliciting a humoral immune response in a human subject.
 6. Thecomposition of claim 1, wherein, when the synthetic nanocarriers areadministered to a human subject, the synthetic nanocarriers are capableof being translocated across a subcapsular sinus (SCS) floor by SCSmacrophages in the subject.
 7. The composition of claim 2, wherein theimmunostimulatory agent is selected from a TLR agonist, an interleukin,an interferon, a cytokine, and an adjuvant.
 8. The composition of claim1, wherein the synthetic nanocarriers comprises polymer molecules, andwherein the plurality of nicotine moieties are covalently attached tothe polymer molecules.
 9. The composition of claim 8, wherein theplurality of nicotine moieties are (S)-(−)-nicotine moieties, andwherein the synthetic nanocarriers comprise a polymer having thestructure of formula (I)(X)_(n)-L1-(Y)_(m)L2-A  (I) wherein: X is a hydrophobic polymer segment;Y is a hydrophilic polymer segment; n and m are selected from 0 and 1,provided that n and m are not both 0; L1 and L2 are independentlyselected from a bond and a linking group; and A is (S)-(−)-nicotine. 10.The composition of claim 8, wherein the plurality of nicotine moietiescomprises (S)-(−)-nicotine having the structure

wherein one of R¹-R⁷ represents the polymer, and the others are selectedfrom H, alkyl, aryl, alkoxy, aryloxy, alkaryl, and aralkyl, any of whichmay be substituted or unsubstituted and may contain one or moreheteroatoms.
 11. The composition of claim 1, wherein the plurality ofnicotine moieties are present in a density equal to or greater than thedensity required to obtain at least 10% of the maximal immobilizationobserved for a monoclonal antibody (MAb) in an antigen presenting cell(APC) binding assay, provided that, in the APC binding assay, the halfmaximal binding density for the plurality of moieties is at least twicethe half maximal binding density for the MAb.
 12. The composition ofclaim 11, wherein the plurality of nicotine moieties are present in adensity equal to or greater than the density required to obtain at least20% of the maximal immobilization observed for a MAb in the APC bindingassay.
 13. The composition of claim 11, wherein the half maximal bindingdensity for the plurality of moieties is at least four times the halfmaximal binding density for the MAb.
 14. The composition of claim 11,wherein the APC binding assay comprises: (a) preparing a series ofsubstrates having coatings of a functional moiety at a series of surfacecoating densities, wherein the functional moiety is capable of bindingto dendritic cell (DC) or subcapsular sinus macrophage surfacereceptors; (b) exposing the series of substrates to single-cellsuspensions of DCs or subcapsular sinus macrophages for a predeterminedperiod of time; (c) removing non-adhered APCs from the series ofsubstrates, and fixing the adhered APCs to the series of substrates; (d)quantifying the number of adhered APCs per unit surface area for eachsubstrate in the series of substrates; (e) plotting the result from (d)against the coating density of the functional moiety; (f) obtaining avalue for the maximal immobilization by determining the maximum numberof adhered APCs per unit surface area for the series of substrates; and(g) obtaining a value for half maximal binding density by determiningthe surface coating density that provides 50% of the maximum.
 15. Thecomposition of claim 11, wherein the MAb is anti-CD1c (BDCA-1) CloneAD5-8E7, or rat anti-mouse CD169, clone 3D6.112, isotype IgG2a.
 16. Thecomposition of claim 1, wherein the nicotine comprises a derivative,metabolite, or analog of nicotine.
 17. A method comprising administeringthe composition of claim 1 to a subject having a baseline IgGanti-nicotine antibody serum concentration of less than 100 ng/ml,wherein a post-administration anti-nicotine IgG antibody peak serumconcentration in the subject is more than 200 ng/ml.
 18. A methodcomprising administering the composition of claim 1 to a subject thathas not been previously vaccinated against nicotine, wherein apost-administration anti-nicotine antibody peak concentration in thesubject is greater than or equal to 100 ng/ml.
 19. A method comprising:administering to a subject an initial dose of the composition of claim1; and administering to the subject a first subsequent dose of thecomposition of claim 1 at a time period after the administration of theinitial dose.
 20. The method of claim 19, wherein the time period afterthe administration of the initial dose is between 1 day and 1 year. 21.The method of claim 19, further comprising administering to the subjecta second subsequent dose of the composition of claim 1 at a time periodafter the administration of the first subsequent dose.
 22. The method ofclaim 21, wherein the time period after the administration of the firstsubsequent dose is between 1 day and 1 year.